Targeted therapeutic proteins

ABSTRACT

Targeted therapeutics that localize to a specific subcellular compartment such as the lysosome are provided. The targeted therapeutics include a therapeutic agent and a targeting moiety that binds a receptor on an exterior surface of the cell, permitting proper subcellular localization of the targeted therapeutic upon internalization of the receptor. Nucleic acids, cells, and methods relating to the practice of the invention are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/483,487, filed Sep. 11, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/920,761, filed Jun. 18, 2013, which is acontinuation of U.S. patent application Ser. No. 13/197,626, filed Aug.3, 2011, which is a continuation of U.S. patent application Ser. No.12/389,235, filed Feb. 19, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/272,483, filed Oct. 16, 2002, now patented asU.S. Pat. No. 7,560,424, which claims the priority benefit of U.S.Provisional Application No. 60/408,816, filed Sep. 6, 2002, U.S.Provisional Application No. 60/386,019, filed Jun. 5, 2002, U.S.Provisional Application No. 60/384,452, filed May 29, 2002, and is acontinuation-in-part of U.S. patent application Ser. No. 10/136,841,filed Apr. 30, 2002, now patented as U.S. Pat. No. 7,396,811, whichclaims the priority benefit of U.S. Provisional Application No.60/351,276, filed Jan. 23, 2002, U.S. Provisional Application No.60/329,461, filed Oct. 15, 2001, U.S. Provisional Application No.60/304,609, filed Jul. 10, 2001, and U.S. Provisional Application No.60/287,531, filed Apr. 30, 2001, the contents of which are herebyincorporated by reference.

This invention provides a means for specifically delivering proteins toa targeted subcellular compartment of a mammalian cell. The ability totarget proteins to a subcellular compartment is of great utility in thetreatment of metabolic diseases such as lysosomal storage diseases, aclass of over 40 inherited disorders in which particular lysosomalenzymes are absent or deficient.

BACKGROUND

Enzyme deficiencies in cellular compartments such as the golgi, theendoplasmic reticulum, and the lysosome cause a wide variety of humandiseases. For example, lysyl hydroxylase, an enzyme normally in thelumen of the endoplasmic reticulum, is required for proper processing ofcollagen; absence of the enzyme causes Ehlers-Danlos syndrome type VI, aserious connective tissue disorder. GnT II, normally found in the golgi,is required for normal glycosylation of proteins; absence of GnT IIcauses leads to defects in brain development. More than forty lysosomalstorage diseases (LSDs) are caused, directly or indirectly, by theabsence of one or more proteins in the lysosome.

Mammalian lysosomal enzymes are synthesized in the cytosol and traversethe ER where they are glycosylated with N-linked, high mannose typecarbohydrate. In the golgi, the high mannose carbohydrate is modified onlysosomal proteins by the addition of mannose-6-phosphate (M6P) whichtargets these proteins to the lysosome. The M6P-modified proteins aredelivered to the lysosome via interaction with either of two M6Preceptors. The most favorable form of modification is when two M6Ps areadded to a high mannose carbohydrate.

Enzyme replacement therapy for lysosomal storage diseases (LSDs) isbeing actively pursued. Therapy, except in Gaucher's disease, generallyrequires that LSD proteins be taken up and delivered to the lysosomes ofa variety of cell types in an M6P-dependent fashion. One possibleapproach involves purifying an LSD protein and modifying it toincorporate a carbohydrate moiety with M6P. This modified material maybe taken up by the cells more efficiently than unmodified LSD proteinsdue to interaction with M6P receptors on the cell surface. However,because of the time and expense required to prepare, purify and modifyproteins for use in subcellular targeting, a need for new, simpler, moreefficient, and more cost-effective methods for targeting therapeuticagents to a cellular compartment remains.

SUMMARY OF THE INVENTION

The present invention facilitates the treatment of metabolic diseases byproviding targeted protein therapeutics that localize to a subcellularcompartment of a cell where the therapeutic is needed. The inventionsimplifies preparation of targeted protein therapeutics by reducingrequirements for posttranslational or postsynthesis processing of theprotein. For example, a targeted therapeutic of the present inventioncan be synthesized as a fusion protein including a therapeutic domainand a domain that targets the fusion protein to a correct subcellularcompartment. (“Fusion protein,” as used herein, refers to a singlepolypeptide having at least two domains that are not normally present inthe same polypeptide. Thus, naturally occurring proteins are not “fusionproteins” as used herein.) Synthesis as a fusion protein permitstargeting of the therapeutic domain to a desired subcellular compartmentwithout complications associated with chemical crosslinking of separatetherapeutic and targeting domains, for example.

The invention also permits targeting of a therapeutic to a lysosome inan M6P-independent manner. Accordingly, the targeted therapeutic neednot be synthesized in a mammalian cell, but can be synthesizedchemically or in a bacterium, yeast, protozoan, or other organismregardless of glycosylation pattern, facilitating production of thetargeted therapeutic with high yield and comparatively low cost. Thetargeted therapeutic can be synthesized as a fusion protein, furthersimplifying production, or can be generated by associatingindependently-synthesized therapeutic agents and targeting moieties.

The present invention permits lysosomal targeting of therapeuticswithout the need for M6P addition to high mannose carbohydrate. It isbased in part on the observation that one of the 2 M6P receptors alsobinds other ligands with high affinity. For example, thecation-independent mannose-6-phosphate receptor is also known as theinsulin-like growth factor 2 (IGF-II) receptor because it binds IGF-IIwith high affinity. This low molecular weight polypeptide interacts withthree receptors, the insulin receptor, the IGF-I receptor and theM6P/IGF-II receptor. It is believed to exert its biological effectprimarily through interactions with the former two receptors whileinteraction with the cation-independent M6P receptor is believed toresult predominantly in the IGF-II being transported to the lysosomewhere it is degraded.

Accordingly, the invention relates in one aspect to a targetedtherapeutic including a targeting moiety and a therapeutic agent that istherapeutically active in a mammalian lysosome. “Therapeuticallyactive,” as used herein, encompasses at least polypeptides or othermolecules that provide an enzymatic activity to a cell or a compartmentthereof that is deficient in that activity. “Therapeutically active”also encompasses other polypeptides or other molecules that are intendedto ameliorate or to compensate for a biochemical deficiency in a cell,but does not encompass molecules that are primarily cytotoxic orcytostatic, such as chemotherapeutics.

In one embodiment, the targeting moiety is a means (e.g. a molecule) forbinding the extracellular domain of the human cation-independent M6Preceptor in an M6P-independent manner when the receptor is present inthe plasma membrane of a target cell. In another embodiment, thetargeting moiety is an unglycosylated lysosomal targeting domain thatbinds the extracellular domain of the human cation-independent M6Preceptor. In either embodiment, the targeting moiety can include, forexample, IGF-II; retinoic acid or a derivative thereof; a protein havingan amino acid sequence at least 70% identical to a domain ofurokinase-type plasminogen activator receptor; an antibody variabledomain that recognizes the receptor; or variants thereof. In someembodiments, the targeting moiety binds to the receptor with asubmicromolar dissociation constant (e.g. less than 10⁻⁸ M, less than10⁻⁹ M, less than 10⁻¹⁰ M, or between 10⁻⁷ M and 10⁻¹¹ M) at or about pH7.4 and with an dissociation constant at or about pH 5.5 of at least10⁻⁶ M and at least ten times the dissociation constant at or about pH7.4. In particular embodiments, the means for binding binds to theextracellular domain at least 10-fold less avidly (i.e. with at least aten-fold greater dissociation constant) at or about pH 5.5 than at orabout pH 7.4; in one embodiment, the dissociation constant at or aboutpH 5.5 is at least 10⁻⁶ M. In a further embodiment, association of thetargeted therapeutic with the means for binding is destabilized by a pHchange from at or about pH 7.4 to at or about pH 5.5.

In another embodiment, the targeting moiety is a lysosomal targetingdomain that binds the extracellular domain of the humancation-independent M6P receptor but does not bind a mutein of thereceptor in which amino acid 1572 is changed from isoleucine tothreonine, or binds the mutein with at least ten-fold less affinity(i.e. with at least a ten-fold greater dissociation constant). Inanother embodiment, the targeting moiety is a lysosomal targeting domaincapable of binding a receptor domain consisting essentially of repeats10-15 of the human cation-independent M6P receptor: the lysosomaltargeting domain can bind a protein that includes repeats 10-15 even ifthe protein includes no other moieties that bind the lysosomal targetingdomain. Preferably, the lysosomal targeting domain can bind a receptordomain consisting essentially of repeats 10-13 of the humancation-independent mannose-6-phosphate receptor. More preferably, thelysosomal targeting domain can bind a receptor domain consistingessentially of repeats 11-12, repeat 11, or amino acids 1508-1566 of thehuman cation-independent M6P receptor. In each of these embodiments, thelysosomal targeting domain preferably binds the receptor or receptordomain with a submicromolar dissociation constant at or about pH 7.4. Inone preferred embodiment, the lysosomal targeting domain binds with andissociation constant of about 10⁻⁷ M. In another preferred embodiment,the dissociation constant is less than about 10⁻⁷ M.

In another embodiment, the targeting moiety is a binding moietysufficiently duplicative of human IGF-II such that the binding moietybinds the human cation-independent M6P receptor. The binding moiety canbe sufficiently duplicative of IGF-II by including an amino acidsequence sufficiently homologous to at least a portion of IGF-II, or byincluding a molecular structure sufficiently representative of at leasta portion of IGF-II, such that the binding moiety binds thecation-independent M6P receptor. The binding moiety can be an organicmolecule having a three-dimensional shape representative of at least aportion of IGF-II, such as amino acids 48-55 of human IGF-II, or atleast three amino acids selected from the group consisting of aminoacids 8, 48, 49, 50, 54, and 55 of human IGF-II. A preferred organicmolecule has a hydrophobic moiety at a position representative of aminoacid 48 of human IGF-II and a positive charge at or about pH 7.4 at aposition representative of amino acid 49 of human IGF-II. In oneembodiment, the binding moiety is a polypeptide including a polypeptidehaving antiparallel alpha-helices separated by not more than five aminoacids. In another embodiment, the binding moiety includes a polypeptidewith the amino acid sequence of IGF-I or of a mutein of IGF-I in whichamino acids 55-56 are changed and/or amino acids 1-4 are deleted orchanged. In a further embodiment, the binding moiety includes apolypeptide with an amino acid sequence at least 60% identical to humanIGF-II; amino acids at positions corresponding to positions 54 and 55 ofhuman IGF-II are preferably uncharged or negatively charged at or aboutpH 7.4.

In one embodiment, the targeting moiety is a polypeptide comprising theamino acid sequence phenylalanine-arginine-serine. In anotherembodiment, the targeting moiety is a polypeptide including an aminoacid sequence at least 75% homologous to amino acids 48-55 of humanIGF-II. In another embodiment, the targeting moiety includes, on asingle polypeptide or on separate polypeptides, amino acids 8-28 and41-61 of human IGF-II. In another embodiment, the targeting moietyincludes amino acids 41-61 of human IGF-II and a mutein of amino acids8-28 of human IGF-II differing from the human sequence at amino acids 9,19, 26, and/or 27.

In some embodiments, the association of the therapeutic agent with thetargeting moiety is labile at or about pH 5.5. In a preferredembodiment, association of the targeting moiety with the therapeuticagent is mediated by a protein acceptor (such as imidazole or aderivative thereof such as histidine) having a pKa between 5.5 and 7.4.Preferably, one of the therapeutic agent or the targeting moiety iscoupled to a metal, and the other is coupled to a pH-dependent metalbinding moiety.

In another aspect, the invention relates to a therapeutic fusion proteinincluding a therapeutic domain and a subcellular targeting domain. Thesubcellular targeting domain binds to an extracellular domain of areceptor on an exterior surface of a cell. Upon internalization of thereceptor, the subcellular targeting domain permits localization of thetherapeutic domain to a subcellular compartment such as a lysosome, anendosome, the endoplasmic reticulum (ER), or the golgi complex, wherethe therapeutic domain is therapeutically active. In one embodiment, thereceptor undergoes constitutive endocytosis. In another embodiment, thetherapeutic domain has a therapeutic enzymatic activity. The enzymaticactivity is preferably one for which a deficiency (in a cell or in aparticular compartment of a cell) is associated with a human diseasesuch as a lysosomal storage disease.

In further aspects, the invention relates to nucleic acids encodingtherapeutic proteins and to cells (e.g. mammalian cells, insect cells,yeast cells, protozoans, or bacteria) comprising these nucleic acids.The invention also provides methods of producing the proteins byproviding these cells with conditions (e.g. in the context of in vitroculture or by maintaining the cells in a mammalian body) permittingexpression of the proteins. The proteins can be harvested thereafter(e.g. if produced in vitro) or can be used without an interveningharvesting step (e.g. if produced in vivo in a patient). Thus, theinvention also provides methods of treating a patient by administering atherapeutic protein (e.g. by injection, in situ synthesis, orotherwise), by administering a nucleic acid encoding the protein(thereby permitting in vivo protein synthesis), or by administering acell comprising a nucleic acid encoding the protein. In one embodiment,the method includes synthesizing a targeted therapeutic including atherapeutic agent that is therapeutically active in a mammalian lysosomeand a targeting moiety that binds human cation-independentmannose-6-phosphate receptor in a mannose-6-phosphate-independentmanner, and administering the targeted therapeutic to a patient. Themethod can also include identifying the targeting moiety (e.g. by arecombinant display technique such as phage display, bacterial display,or yeast two-hybrid or by screening libraries for requisite bindingproperties). In another embodiment, the method includes providing (e.g.on a computer) a molecular model defining a three-dimensional shaperepresentative of at least a portion of human IGF-II; identifying acandidate IGF-II analog having a three-dimensional shape representativeof at least a portion of IGF-II (e.g. amino acids 48-55), and producinga therapeutic agent that is active in a mammalian lysosome and directlyor indirectly bound to the candidate IGF-II analog. The method can alsoinclude determining whether the candidate IGF-II analog binds to thehuman cation-independent M6P receptor.

This invention also provides methods for producing therapeutic proteinsthat are targeted to lysosomes and/or across the blood-brain barrier andthat possess an extended half-life in circulation in a mammal. Themethods include producing an underglycosylated therapeutic protein. Asused herein, “underglycosylated” refers to a protein in which one ormore carbohydrate structures that would normally be present if theprotein were produced in a mammalian cell (such as a CHO cell) has beenomitted, removed, modified, or masked, thereby extending the half-lifeof the protein in a mammal. Thus, a protein may be actuallyunderglycosylated due to the absence of one or more carbohydratestructures, or functionally underglycosylated by modification or maskingof one or more carbohydrate structures that promote clearance fromcirculation. For example, a structure could be masked (i) by theaddition of one or more additional moieties (e.g. carbohydrate groups,phosphate groups, alkyl groups, etc.) that interfere with recognition ofthe structure by a mannose or asialoglycoprotein receptor, (ii) bycovalent or noncovalent association of the glycoprotein with a bindingmoiety, such as a lectin or an extracellular portion of a mannose orasialoglycoprotein receptor, that interferes with binding to thosereceptors in vivo, or (iii) any other modification to the polypeptide orcarbohydrate portion of a glycoprotein to reduce its clearance from theblood by masking the presence of all or a portion of the carbohydratestructure.

In one embodiment, the therapeutic protein includes a peptide targetingmoiety (e.g. IGF-I, IGF-II, or a portion thereof effective to bind atarget receptor) that is produced in a host (e.g. bacteria or yeast)that does not glycosylate proteins as conventional mammalian cells (e.g.Chinese hamster ovary (CHO) cells) do. For example, proteins produced bythe host cell may lack terminal mannose, fucose, and/orN-acetylglucosamine residues, which are recognized by the mannosereceptor, or may be completely unglycosylated. In another embodiment,the therapeutic protein, which may be produced in mammalian cells or inother hosts, is treated chemically or enzymatically to remove one ormore carbohydrate residues (e.g. one or more mannose, fucose, and/orN-acetylglucosamine residues) or to modify or mask one or morecarbohydrate residues. Such a modification or masking may reduce bindingof the therapeutic protein to the hepatic mannose and/orasialoglycoprotein receptors. In another embodiment, one or morepotential glycosylation sites are removed by mutation of the nucleicacid encoding the targeted therapeutic protein, thereby reducingglycosylation of the protein when synthesized in a mammalian cell orother cell that glycosylates proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts several types of underglycosylation.

FIGS. 2A-2B are a map of the human IGF-II open reading frame (SEQ IDNO:1) and its encoded protein (SEQ ID NO:2). Mature IGF-II lacks thesignal peptide and COOH-cleaved regions.

FIG. 3 is a Leishmania codon-optimized IGF-II depicted in the XbaI siteof pIR1-SAT; the nucleic acid is SEQ ID NO:3 and the encoded protein isSEQ ID NO:4.

FIGS. 4A-4E are a depiction of a preferred embodiment of the invention,incorporating a signal peptide sequence, the mature humanβ-glucuronidase sequence, a bridge of three amino acids, and an IGF-IIsequence. The depicted nucleic acid is SEQ ID NO:5, and the encodedprotein is SEQ ID NO:6.

FIG. 5 depicts β-glucuronidase (GUS) activity in humanmucopolysaccharidosis VII skin fibroblast GM4668 cells exposed to GUS, aGUS-IGF-II fusion protein (GILT-GUS), GILT-GUS with Δ1-7 and Y27Lmutations in the IGF-II portion (GILT²-GUS), or a negative control(DMEM).

FIG. 6 depicts GUS activity in GM4668 cells exposed to GUS (+β-GUS),GUS-GILT (+GILT), GUS-GILT in the presence of excess IGF-II(+GILT+IGF-II), or a negative control (GM4668).

FIG. 7 is an alignment of human IGF-I (SEQ ID NO:7) and IGF-II (SEQ IDNO:8), showing the A, B, C, and D domains.

FIG. 8 depicts GUS in GM4668 cells exposed to GUS, GUS-GILT, GUS-GILT,GUS-GILT with a deletion of the seven amino-terminal residues (GUS-GILTΔ1-7), GUS-GILT in the presence of excess IGF-II, GUS-GILT Δ1-7 in thepresence of excess IGF-II, or a negative control (Mock).

FIG. 9A depicts one form of a phosphorylated high mannose carbohydratestructure linked to a glycoprotein via an asparagine residue, and alsodepicts the structures of mannose and N-acetylglucosamine (GlcNAc). FIG.9B depicts a portion of the high mannose carbohydrate structure at ahigher level of detail, and indicates positions vulnerable to cleavageby periodate treatment. The positions of the sugar residues within thecarbohydrate structure are labeled with circled, capital letters A-H;phosphate groups are indicated with a circled capital P.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “glycosylation independent lysosomal targeting” and“GILT” refer to lysosomal targeting that ismannose-6-phosphate-independent.

As used herein, “GILT construct” refers to a construct including amannose-6-phosphate-independent lysosomal targeting portion and atherapeutic portion effective in a mammalian lysosome.

As used herein, “GUS” refers to β-glucuronidase, an exemplarytherapeutic portion.

As used herein, “GUSΔC18” refers to GUS with a deletion of theC-terminal 18 amino acids, removing a potential proteolysis site.

As used herein, “GUS-GILT” refers to a GILT construct with GUS coupledto an IGF-II targeting portion.

All references to amino acid positions in refer to the positions inmature human IGF-II. Thus, for example, positions 1, 2, and 3 areoccupied by alanine, tyrosine, and arginine, respectively.

As used herein, GILTΔ1-7 refers to an IGF-II targeting portion with adeletion of the N-terminal 7 amino acids.

As used herein, GUSΔC18-GILTΔ1-7 refers to a fusion protein in whichGUSΔC18 is fused to the N-terminus of GILTΔ1-7.

The present invention facilitates treatment of metabolic diseases byproviding targeted therapeutics that, when provided externally to acell, enter the cell and localize to a subcellular compartment where thetargeted therapeutic is active. The targeted therapeutic includes atleast a therapeutic agent and a targeting moiety, such as a subcellulartargeting domain of a protein, or, for lysosomal targeting, a means(e.g. a protein, peptide, peptide analog, or organic chemical) forbinding the human cation-independent mannose-6-phosphate receptor.

Association Between Therapeutic Agent and Targeting Moiety

The therapeutic agent and the targeting moiety are necessarilyassociated, directly or indirectly. In one embodiment, the therapeuticagent and the targeting moiety are non-covalently associated. Theassociation is preferably stable at or about pH 7.4. For example, thetargeting moiety can be biotinylated and bind avidin associated with thetherapeutic agent. Alternatively, the targeting moiety and thetherapeutic agent can each be associated (e.g. as fusion proteins) withdifferent subunits of a multimeric protein. In another embodiment, thetargeting moiety and the therapeutic agent are crosslinked to each other(e.g. using a chemical crosslinking agent).

In a preferred embodiment, the therapeutic agent is fused to thetargeting moiety as a fusion protein. The targeting moiety can be at theamino-terminus of the fusion protein, the carboxy-terminus, or can beinserted within the sequence of the therapeutic agent at a positionwhere the presence of the targeting moiety does not unduly interferewith the therapeutic activity of the therapeutic agent.

Where the therapeutic agent is a heteromeric protein, one or more of thesubunits can be associated with a targeting portion. Hexosaminidase A,for example, a lysosomal protein affected in Tay-Sachs disease, includesan alpha subunit and a beta subunit. The alpha subunit, the betasubunit, or both can be associated with a targeting moiety in accordancewith the present invention. If, for example, the alpha subunit isassociated with a targeting moiety and is coexpressed with the betasubunit, an active complex is formed and targeted appropriately (e.g. tothe lysosome).

For targeting a therapeutic to the lysosome, the therapeutic agent canbe connected to the targeting moiety through an interaction that isdisrupted by decreasing the pH from at or about 7.4 to at or about 5.5.The targeting moiety binds a receptor on the exterior of a cell; theselected receptor is one that undergoes endocytosis and passes throughthe late endosome, which has a pH of about 5.5. Thus, in the lateendosome, the therapeutic agent dissociates from the targeting moietyand proceeds to the lysosome, where the therapeutic agent acts. Forexample, a targeting moiety can be chemically modified to incorporate achelating agent (e.g. EDTA, EGTA, or trinitrilotriacetic acid) thattightly binds a metal ion such as nickel. The targeting moiety (e.g.GUS) can be expressed as a fusion protein with a six-histidine tag (e.g.at the amino-terminus, at the carboxy-terminus, or in asurface-accessible flexible loop). At or about pH 7.4, the six-histidinetag is substantially deprotonated and binds metal ions such as nickelwith high affinity. At or about pH 5.5, the six-histidine tag issubstantially protonated, leading to release of the nickel and,consequently, release of the therapeutic agent from the targetingmoiety.

Therapeutic Agent

While methods and compositions of the invention are useful for producingand delivering any therapeutic agent to a subcellular compartment, theinvention is particularly useful for delivering gene products fortreating metabolic diseases.

Preferred LSD genes are shown in Table 1, and preferred genes associatedwith golgi or ER defects are shown in Table 2. In a preferredembodiment, a wild-type LSD gene product is delivered to a patientsuffering from a defect in the same LSD gene. In alternativeembodiments, a functional sequence or species variant of the LSD gene isused. In further embodiments, a gene coding for a different enzyme thatcan rescue an LSD gene defect is used according to methods of theinvention.

TABLE 1 Lysosomal Storage Diseases and associated enzyme defectsSubstance Disease Name Enzyme Defect Stored A. Glycogenosis DisordersPompe Disease Acid-a1,4- Glycogen α 1-4 linked GlucosidaseOligosaccharides B. Glycolipidosis Disorders GM1 Gangliodsidosisβ-Galactosidase GM₁ Ganliosides Tay-Sachs Disease β-Hexosaminidase A GM₂Ganglioside GM2 Gangliosidosis: GM₂ Activator GM₂ Ganglioside AB VariantProtein Sandhoff Disease β-Hexosaminidase GM₂ Ganglioside A&B FabryDisease α-Galactosidase A Globosides Gaucher Disease GlucocerebrosidaseGlucosylceramide Metachromatic Arylsulfatase A SulphatidesLeukodystrophy Krabbe Disease Galactosylceramidase GalactocerebrosideNiemann-Pick, Types Acid Sphingomyelin A and B SphingomyelinaseNiemann-Pick, Type C Cholesterol Sphingomyelin Esterification DefectNieman-Pick, Type D Unknown Sphingomyelin Farber Disease Acid CeramidaseCeramide Wolman Disease Acid Lipase Cholesteryl Esters C.Mucopolysaccharide Disorders Hurler Syndrome α-L-Iduronidase Heparan &(MPS IH) Dermatan Sulfates Scheie Syndrome α-L-Iduronidase Heparan &(MPS IS) Dermatan, Sulfates Hurler-Scheie α-L-Iduronidase Heparan & (MPSIH/S) Dermatan Sulfates Hunter Syndrome Iduronate Sulfatase Heparan &(MPS II) Dermatan Sulfates Sanfilippo A Heparan N-Sulfatase Heparan (MPSIIIA) Sulfate Sanfilippo B α-N- Heparan (MPS IIIB) AcetylglucosaminidaseSulfate Sanfilippo C Acetyl-CoA- Heparan (MPS IIIC) GlucosaminideSulfate Acetyltransferase Sanfilippo D N-Acetylglucosamine- Heparan (MPSIIID) 6-Sulfatase Sulfate Morquio A Galactosamine-6- Keratan (MPS IVA)Sulfatase Sulfate Morquio B β-Galactosidase Keratan (MPS IVB) SulfateMaroteaux-Lamy Arylsulfatase B Dermatan (MPS VI) Sulfate Sly Syndromeβ-Glucuronidase (MPS VII) D. Oligosaccharide/Glycoprotein Disordersα-Mannosidosis α-Mannosidase Mannose/ Oligosaccharides β-Mannosidosisβ-Mannosidase Mannose/ Oligosaccharides Fucosidosis α-L-FucosidaseFucosyl Oligosaccharides Asparylglucosaminuria N-Aspartyl-β-Asparylglucosamine Glucosaminidase Asparagines Sialidosisα-Neuraminidase Sialyloligosaccharides (Mucolipidosis I)Galactosialidosis Lysosomal Protective Sialyloligosaccharides (GoldbergSyndrome) Protein Deficiency Schindler Disease α-N-Acetyl-Galactosaminidase E. Lysosomal Enzyme Transport Disorders MucolipidosisII (I- N-Acetylglucosamine- Heparan Sulfate Cell Disease)1-Phosphotransferase Mucolipidosis III Same as ML II (Pseudo-HurlerPolydystrophy) F. Lysosomal Membrane Transport Disorders CystinosisCystine Transport Free Cystine Protein Salla Disease Sialic AcidTransport Free Sialic Acid and Protein Glucuronic Acid Infantile SialicAcid Sialic Acid Transport Free Sialic Acid and Storage Disease ProteinGlucuronic Acid G. Other Batten Disease Unknown Lipofuscins (JuvenileNeuronal Ceroid Lipofuscinosis) Infantile Neuronal Palmitoyl-ProteinLipofuscins Ceroid Lipofuscinosis Thioesterase Mucolipidosis IV UnknownGangliosides & Hyaluronic Acid Prosaposin Saposins A, B, C or D

TABLE 2 Diseases of the golgi and ER Disease Name Gene and Enzyme DefectFeatures Ehlers-Danlos Syndrome Type PLOD1 lysyl hydroxylase Defect inlysyl hydroxylation VI of Collagen; located in ER lumen Type Ia glycogestorage glucose6 phosphatase Causes excessive disease accumulation ofGlycogen in the liver, kidney, and Intestinal mucosa; enzyme istransmembrane but active site is ER lumen Congenital Disorders ofGlycosylation CDG Ic ALG6 Defects in N-glycosylation ER α1,3glucosyltransferase lumen CDG Id ALG3 Defects in N-glycosylation ER α1,3mannosyltransferase transmembrane protein CDG IIa MGAT2 Defects inN-glycosylation N-acetylglucosaminyl- golgi transmembrane proteintransferase II CDG IIIb GCS1 Defect in N glycosylation α1,2-GlucosidaseI ER membrane bound with lumenal catalytic domain releasable byproteolysis

One particularly preferred therapeutic agent is glucocerebrosidase,currently manufactured by Genzyme as an effective enzyme replacementtherapy for Gaucher's Disease. Currently, the enzyme is prepared withexposed mannose residues, which targets the protein specifically tocells of the macrophage lineage. Although the primary pathology in type1 Gaucher patients are due to macrophage accumulating glucocerebroside,there can be therapeutic advantage to delivering glucocerebrosidase toother cell types. Targeting glucocerebrosidase to lysosomes using thepresent invention would target the agent to multiple cell types and canhave a therapeutic advantage compared to other preparations.

Subcellular Targeting Domains

The present invention permits targeting of a therapeutic agent to alysosome using a protein, or an analog of a protein, that specificallybinds a cellular receptor for that protein. The exterior of the cellsurface is topologically equivalent to endosomal, lysosomal, golgi, andendoplasmic reticulum compartments. Thus, endocytosis of a moleculethrough interaction with an appropriate receptor(s) permits transport ofthe molecule to any of these compartments without crossing a membrane.Should a genetic deficiency result in a deficit of a particular enzymeactivity in any of these compartments, delivery of a therapeutic proteincan be achieved by tagging it with a ligand for the appropriatereceptor(s).

Multiple pathways directing receptor-bound proteins from the plasmamembrane to the golgi and/or endoplasmic reticulum have beencharacterized. Thus, by using a targeting portion from, for example,SV40, cholera toxin, or the plant toxin ricin, each of which coopt oneor more of these subcellular trafficking pathways, a therapeutic can betargeted to the desired location within the cell. In each case, uptakeis initiated by binding of the material to the exterior of the cell. Forexample, SV40 binds to MHC class I receptors, cholera toxin binds to GM1ganglioside molecules and ricin binds to glycolipids and glycoproteinswith terminal galactose on the surface of cells. Following this initialstep the molecules reach the ER by a variety of pathways. For example,SV40 undergoes caveolar endocytosis and reaches the ER in a two stepprocess that bypasses the golgi whereas cholera toxin undergoes caveolarendocytosis but traverses the golgi before reaching the ER.

If a targeting moiety related to cholera toxin or ricin is used, it isimportant that the toxicity of cholera toxin or ricin be avoided. Bothcholera toxin and ricin are heteromeric proteins, and the cell surfacebinding domain and the catalytic activities responsible for toxicityreside on separate polypeptides. Thus, a targeting moiety can beconstructed that includes the receptor-binding polypeptide, but not thepolypeptide responsible for toxicity. For example, in the case of ricin,the B subunit possesses the galactose binding activity responsible forinternalization of the protein, and can be fused to a therapeuticprotein. If the further presence of the A subunit improves subcellularlocalization, a mutant version (mutein) of the A chain that is properlyfolded but catalytically inert can be provided with the Bsubunit-therapeutic agent fusion protein.

Proteins delivered to the golgi can be transported to the endoplasmicreticulum (ER) via the KDEL receptor, which retrieves ER-targetedproteins that have escaped to the golgi. Thus, inclusion of a KDEL motifat the terminus of a targeting domain that directs a therapeutic proteinto the golgi permits subsequent localization to the ER. For example, atargeting moiety (e.g. an antibody, or a peptide identified byhigh-throughput screening such as phage display, yeast two hybrid,chip-based assays, and solution-based assays) that binds thecation-independent M6P receptor both at or about pH 7.4 and at or aboutpH 5.5 permits targeting of a therapeutic agent to the golgi; furtheraddition of a KDEL motif permits targeting to the ER.

Lysosomal Targeting Moieties

The invention permits targeting of a therapeutic agent to a lysosome.Targeting may occur, for example, through binding of a plasma membranereceptor that later passes through a lysosome. Alternatively, targetingmay occur through binding of a plasma receptor that later passes througha late endosome; the therapeutic agent can then travel from the lateendosome to a lysosome. A preferred lysosomal targeting mechanisminvolves binding to the cation-independent M6P receptor.

Cation-independent M6P Receptor

The cation-independent M6P receptor is a 275 kDa single chaintransmembrane glycoprotein expressed ubiquitously in mammalian tissues.It is one of two mammalian receptors that bind M6P: the second isreferred to as the cation-dependent M6P receptor. The cation-dependentM6P receptor requires divalent cations for M6P binding; thecation-independent M6P receptor does not. These receptors play animportant role in the trafficking of lysosomal enzymes throughrecognition of the M6P moiety on high mannose carbohydrate on lysosomalenzymes. The extracellular domain of the cation-independent M6P receptorcontains 15 homologous domains (“repeats”) that bind a diverse group ofligands at discrete locations on the receptor.

The cation-independent M6P receptor contains two binding sites for M6P:one located in repeats 1-3 and the other located in repeats 7-9. Thereceptor binds monovalent M6P ligands with a dissociation constant inthe μM range while binding divalent M6P ligands with a dissociationconstant in the nM range, probably due to receptor oligomerization.Uptake of IGF-II by the receptor is enhanced by concomitant binding ofmultiyalent M6P ligands such as lysosomal enzymes to the receptor.

The cation-independent M6P receptor also contains binding sites for atleast three distinct ligands that can be used as targeting moieties. Thecation-independent M6P receptor binds IGF-II with a dissociationconstant of about 14 nM at or about pH 7.4, primarily throughinteractions with repeat 11. Consistent with its function in targetingIGF-II to the lysosome, the dissociation constant is increasedapproximately 100-fold at or about pH 5.5 promoting dissociation ofIGF-II in acidic late endosomes. The receptor is capable of binding highmolecular weight O-glycosylated IGF-II forms.

An additional useful ligand for the cation-independent M6P receptor isretinoic acid. Retinoic acid binds to the receptor with a dissociationconstant of 2.5 nM. Affinity photolabeling of the cation-independent M6Preceptor with retinoic acid does not interfere with IGF-II or M6Pbinding to the receptor, indicating that retinoic acid binds to adistinct site on the receptor. Binding of retinoic acid to the receptoralters the intracellular distribution of the receptor with a greateraccumulation of the receptor in cytoplasmic vesicles and also enhancesuptake of M6P modified β-glucuronidase. Retinoic acid has aphotoactivatable moiety that can be used to link it to a therapeuticagent without interfering with its ability to bind to thecation-independent M6P receptor.

The cation-independent M6P receptor also binds the urokinase-typeplasminogen receptor (uPAR) with a dissociation constant of 9 μM. uPARis a GPI-anchored receptor on the surface of most cell types where itfunctions as an adhesion molecule and in the proteolytic activation ofplasminogen and TGF-β. Binding of uPAR to the CI-M6P receptor targets itto the lysosome, thereby modulating its activity. Thus, fusing theextracellular domain of uPAR, or a portion thereof competent to bind thecation-independent M6P receptor, to a therapeutic agent permitstargeting of the agent to a lysosome.

IGF-II

In a preferred embodiment, the lysosomal targeting portion is a protein,peptide, or other moiety that binds the cation independent M6P/IGF-IIreceptor in a mannose-6-phosphate-independent manner. Advantageously,this embodiment mimics the normal biological mechanism for uptake of LSDproteins, yet does so in a manner independent of mannose-6-phosphate.

For example, by fusing DNA encoding the mature IGF-II polypeptide to the3′ end of LSD gene cassettes, fusion proteins are created that can betaken up by a variety of cell types and transported to the lysosome.Alternatively, DNA encoding a precursor IGF-II polypeptide can be fusedto the 3′ end of an LSD gene cassette; the precursor includes acarboxyterminal portion that is cleaved in mammalian cells to yield themature IGF-II polypeptide, but the IGF-II signal peptide is preferablyomitted (or moved to the 5′ end of the LSD gene cassette). This methodhas numerous advantages over methods involving glycosylation includingsimplicity and cost effectiveness, because once the protein is isolated,no further modifications need be made.

IGF-II is preferably targeted specifically to the M6P receptor.Particularly useful are mutations in the IGF-II polypeptide that resultin a protein that binds the M6P receptor with high affinity while nolonger binding the other two receptors with appreciable affinity. IGF-IIcan also be modified to minimize binding to serum IGF-binding proteins(Baxter (2000) Am. J. Physiol Endocrinol Metab. 278(6):967-76) to avoidsequestration of IGF-II/GILT constructs. A number of studies havelocalized residues in IGF-I and IGF-II necessary for binding toIGF-binding proteins. Constructs with mutations at these residues can bescreened for retention of high affinity binding to the M6P/IGF-IIreceptor and for reduced affinity for IGF-binding proteins. For example,replacing PHE 26 of with SER is reported to reduce affinity of IGF-IIfor IGFBP-1 and -6 with no effect on binding to the M6P/IGF-II receptor(Bach et al (1993) J. Biol. Chem. 268(13):9246-54). Other substitutions,such as SER for PHE 19 and LYS for GLU 9, can also be advantageous. Theanalogous mutations, separately or in combination, in a region of IGF-Ithat is highly conserved with IGF-II result in large decreases in IGF-BPbinding (Magee et al. (1999) Biochemistry 38(48):15863-70).

An alternate approach is to identify minimal regions of IGF-II that canbind with high affinity to the M6P/IGF-II receptor. The residues thathave been implicated in IGF-II binding to the M6P/IGF-II receptor mostlycluster on one face of IGF-II (Terasawa et al. (1994) EMBO J.13(23):5590-7). Although IGF-II tertiary structure is normallymaintained by three intramolecular disulfide bonds, a peptideincorporating the amino acid sequence on the M6P/IGF-II receptor bindingsurface of IGF-II can be designed to fold properly and have bindingactivity. Such a minimal binding peptide is a highly preferred targetingportion. Designed peptides based on the region around amino acids 48-55can be tested for binding to the M6P/IGF-II receptor. Alternatively, arandom library of peptides can be screened for the ability to bind theM6P/IGF-II receptor either via a yeast two hybrid assay, or via a phagedisplay type assay.

Blood Brain Barrier

One challenge in therapy for lysosomal storage diseases is that many ofthese diseases have significant neurological involvement. Therapeuticenzymes administered into the blood stream generally do not cross theblood brain barrier and therefore cannot relieve neurological symptomsassociated with the diseases. IGF-II, however, has been reported topromote transport across the blood brain barrier via transcytosis(Bickel et al. (2001) Adv. Drug Deliv. Rev, 46(1-3):247-79). Thus,appropriately designed GILT constructs should be capable of crossing theblood brain barrier, affording for the first time a means of treatingneurological symptoms associated with lysosomal storage diseases. Theconstructs can be tested using GUS minus mice as described in Example12. Further details regarding design, construction and testing oftargeted therapeutics that can reach neuronal tissue from blood aredisclosed in U.S. Ser. No. 60/329,650, filed Oct. 16, 2001, and in U.S.Ser. No. 10/136,639, filed Apr. 30, 2002.

Structure of IGF-II

NMR structures of IGF-II have been solved by two groups (Terasawa et al.(1994) EMBO J. 13(23):5590-7; Torres et al. (1995) J. Mol. Biol.248(2):385-401) (see, e.g., Protein Data Bank record 1IGL). The generalfeatures of the IGF-II structure are similar to IGF-I and insulin. The Aand B domains of IGF-II correspond to the A and B chains of insulin.Secondary structural features include an alpha helix from residues 11-21of the B region connected by a reverse turn in residues 22-25 to a shortbeta strand in residues 26-28. Residues 25-27 appear to form a smallantiparallel beta sheet; residues 59-61 and residues 26-28 may alsoparticipate in intermolecular beta-sheet formation. In the A domain ofalpha helices spanning residues 42-49 and 53-59 are arranged in anantiparallel configuration perpendicular to the B-domain helix.Hydrophobic clusters formed by two of the three disulfide bridges andconserved hydrophobic residues stabilize these secondary structurefeatures. The N and C termini remain poorly defined as is the regionbetween residues 31-40.

IGF-II binds to the IGF-II/M6P and IGF-I receptors with relatively highaffinity and binds with lower affinity to the insulin receptor. IGF-IIalso interacts with a number if serum IGFBPs.

Binding to the IGF-II/M6P Receptor

Substitution of IGF-II residues 48-50 (Phe Arg Ser) with thecorresponding residues from insulin, (Thr Ser Ile), or substitution ofresidues 54-55 (Ala Leu) with the corresponding residues from IGF-I (ArgArg) result in diminished binding to the IGF-II/M6P receptor butretention of binding to the IGF-I and insulin receptors (Sakano et al.(1991) J. Biol. Chem. 266(31):20626-35).

IGF-I and IGF-II share identical sequences and structures in the regionof residues 48-50 yet have a 1000-fold difference in affinity for theIGF-II receptor. The NMR structure reveals a structural differencebetween IGF-I and IGF-II in the region of IGF-II residues 53-58 (IGF-Iresidues 54-59): the alpha-helix is better defined in IGF-II than inIGF-I and, unlike IGF-I, there is no bend in the backbone aroundresidues 53 and 54 (Torres et al. (1995) J. Mol. Biol. 248(2):385-401).This structural difference correlates with the substitution of Ala 54and Leu 55 in IGF-II with Arg 55 and Arg 56 in IGF-I. It is possibleeither that binding to the IGF-II receptor is disrupted directly by thepresence of charged residues in this region or that changes in thestructure engendered by the charged residues yield the changes inbinding for the IGF-II receptor. In any case, substitution of unchargedresidues for the two Arg residues in IGF-I resulted in higher affinitiesfor the IGF-II receptor (Cacciari et al. (1987) Pediatrician14(3):146-53). Thus the presence of positively charged residues in thesepositions correlates with loss of binding to the IGF-II receptor.

IGF-II binds to repeat 11 of the cation-independent M6P receptor.Indeed, a minireceptor in which only repeat 11 is fused to thetransmembrane and cytoplasmic domains of the cation-independent M6Preceptor is capable of binding IGF-II (with an affinity approximatelyone tenth the affinity of the full length receptor) and mediatinginternalization of IGF-II and its delivery to lysosomes (Grimme et al.(2000) J. Biol. Chem. 275(43):33697-33703). The structure of domain 11of the M6P receptor is known (Protein Data Base entries 1GP0 and 1GP3;Brown et al. (2002) EMBO J. 21(5):1054-1062). The putative IGF-IIbinding site is a hydrophobic pocket believed to interact withhydrophobic amino acids of IGF-II; candidate amino acids of IGF-IIinclude leucine 8, phenylalanine 48, alanine 54, and leucine 55.Although repeat 11 is sufficient for IGF-II binding, constructsincluding larger portions of the cation-independent M6P receptor (e.g.repeats 10-13, or 1-15) generally bind IGF-II with greater affinity andwith increased pH dependence (see, for example, Linnell et al. (2001) J.Biol. Chem. 276(26):23986-23991).

Binding to the IGF-I Receptor

Substitution of IGF-II residues Tyr 27 with Leu, Leu 43 with Val or Ser26 with Phe diminishes the affinity of IGF-II for the IGF-I receptor by94-, 56-, and 4-fold respectively (Torres et al. (1995) J. Mol. Biol.248(2):385-401). Deletion of residues 1-7 of human IGF-II resulted in a30-fold decrease in affinity for the human IGF-I receptor and aconcomitant 12 fold increase in affinity for the rat IGF-II receptor(Hashimoto et al. (1995) J. Biol. Chem, 270(30):18013-8). The NMRstructure of IGF-II shows that Thr 7 is located near residues 48 Phe and50 Ser as well as near the 9 Cys-47 Cys disulfide bridge. It is thoughtthat interaction of Thr 7 with these residues can stabilize the flexibleN-terminal hexapeptide required for IGF-I receptor binding (Terasawa etal. (1994) EMBO J. 13(23)5590-7). At the same time this interaction canmodulate binding to the IGF-II receptor. Truncation of the C-terminus ofIGF-II (residues 62-67) also appear to lower the affinity of IGF-II forthe IGF-I receptor by 5 fold (Roth et al. (1991) Biochem. Biophys. Res.Commun. 181(2):907-14).

Deletion Mutants of IGF-II

The binding surfaces for the IGF-I and cation-independent M6P receptorsare on separate faces of IGF-II. Based on structural and mutationaldata, functional cation-independent M6P binding domains can beconstructed that are substantially smaller than human IGF-II. Forexample, the amino terminal amino acids 1-7 and/or the carboxy terminalresidues 62-67 can be deleted or replaced. Additionally, amino acids29-40 can likely be eliminated or replaced without altering the foldingof the remainder of the polypeptide or binding to the cation-independentM6P receptor. Thus, a targeting moiety including amino acids 8-28 and41-61 can be constructed. These stretches of amino acids could perhapsbe joined directly or separated by a linker. Alternatively, amino acids8-28 and 41-61 can be provided on separate polypeptide chains.Comparable domains of insulin, which is homologous to IGF-II and has atertiary structure closely related to the structure of IGF-II, havesufficient structural information to permit proper refolding into theappropriate tertiary structure, even when present in separatepolypeptide chains (Wang et al. (1991) Trends Biochem. Sci. 279-281).Thus, for example, amino acids 8-28, or a conservative substitutionvariant thereof, could be fused to a therapeutic agent; the resultingfusion protein could be admixed with amino acids 41-61, or aconservative substitution variant thereof, and administered to apatient.

Binding to IGF Binding Proteins

IGF-II and related constructs can be modified to diminish their affinityfor IGFBPs, thereby increasing the bioavailability of the taggedproteins.

Substitution of residue phenylalanine 26 with serine reduces binding toIGFBPs 1-5 by 5-75 fold (Bach et al. (1993) J. Biol. Chem.268(13):9246-54). Replacement of IGF-II residues 48-50 withthreonine-serine-isoleucine reduces binding by more than 100 fold tomost of the IGFBPs (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54);these residues are, however, also important for binding to thecation-independent mannose-6-phosphate receptor. The Y27L substitutionthat disrupts binding to the IGF-I receptor interferes with formation ofthe ternary complex with IGFBP3 and acid labile subunit (Hashimoto etal. (1997) J. Biol. Chem. 272(44):27936-42); this ternary complexaccounts for most of the IGF-II in the circulation (Yu et al. (1999) J.Clin. Lab Anal. 13(4):166-72). Deletion of the first six residues ofIGF-II also interferes with IGFBP binding (Luthi et al. (1992) Eur. J.Biochem. 205(2):483-90).

Studies on IGF-I interaction with IGFBPs revealed additionally thatsubstitution of serine for phenylalanine 16 did not effect secondarystructure but decreased IGFBP binding by between 40 and 300 fold (Mageeet al. (1999) Biochemistry 38(48):15863-70). Changing glutamate 9 tolysine also resulted in a significant decrease in IGFBP binding.Furthermore, the double mutant lysine 9/serine 16 exhibited the lowestaffinity for IGFBPs. Although these mutations have not previously beentested in IGF-II, the conservation of sequence between this region ofIGF-I and IGF-II suggests that a similar effect will be observed whenthe analogous mutations are made in IGF-II (glutamate 12lysine/phenylalanine 19 serine).

IGF-II Homologs

The amino acid sequence of human IGF-II, or a portion thereof affectingbinding to the cation-independent M6P receptor, may be used as areference sequence to determine whether a candidate sequence possessessufficient amino acid similarity to have a reasonable expectation ofsuccess in the methods of the present invention. Preferably, variantsequences are at least 70% similar or 60% identical, more preferably atleast 75% similar or 65% identical, and most preferably 80% similar or70% identical to human IGF-II.

To determine whether a candidate peptide region has the requisitepercentage similarity or identity to human IGF-II, the candidate aminoacid sequence and human IGF-II are first aligned using the dynamicprogramming algorithm described in Smith and Waterman (1981) J. Mol.Biol. 147:195-197, in combination with the BLOSUM62 substitution matrixdescribed in FIG. 2 of Henikoff and Henikoff (1992) PNAS 89:10915-10919.For the present invention, an appropriate value for the gap insertionpenalty is −12, and an appropriate value for the gap extension penaltyis −4. Computer programs performing alignments using the algorithm ofSmith-Waterman and the BLOSUM62 matrix, such as the GCG program suite(Oxford Molecular Group, Oxford, England), are commercially availableand widely used by those skilled in the art.

Once the alignment between the candidate and reference sequence is made,a percent similarity score may be calculated. The individual amino acidsof each sequence are compared sequentially according to their similarityto each other. If the value in the BLOSUM62 matrix corresponding to thetwo aligned amino acids is zero or a negative number, the pairwisesimilarity score is zero; otherwise the pairwise similarity score is1.0. The raw similarity score is the sum of the pairwise similarityscores of the aligned amino acids. The raw score is then normalized bydividing it by the number of amino acids in the smaller of the candidateor reference sequences. The normalized raw score is the percentsimilarity. Alternatively, to calculate a percent identity, the alignedamino acids of each sequence are again compared sequentially. If theamino acids are non-identical, the pairwise identity score is zero;otherwise the pairwise identity score is 1.0. The raw identity score isthe sum of the identical aligned amino acids. The raw score is thennormalized by dividing it by the number of amino acids in the smaller ofthe candidate or reference sequences. The normalized raw score is thepercent identity. Insertions and deletions are ignored for the purposesof calculating percent similarity and identity. Accordingly, gappenalties are not used in this calculation, although they are used inthe initial alignment.

IGF-II Structural Analogs

The known structures of human IGF-II and the cation-independent M6Preceptors permit the design of IGF-II analogs and othercation-independent M6P receptor binding proteins using computer-assisteddesign principles such as those discussed in U.S. Pat. Nos. 6,226,603and 6,273,598. For example, the known atomic coordinates of IGF-II canbe provided to a computer equipped with a conventional computer modelingprogram, such as INSIGHTII, DISCOVER, or DELPHI, commercially availablefrom Biosym, Technologies Inc., or QUANTA, or CHARMM, commerciallyavailable from Molecular Simulations, Inc. These and other softwareprograms allow analysis of molecular structures and simulations thatpredict the effect of molecular changes on structure and onintermolecular interactions. For example, the software can be used toidentify modified analogs with the ability to form additionalintermolecular hydrogen or ionic bonds, improving the affinity of theanalog for the target receptor.

The software also permits the design of peptides and organic moleculeswith structural and chemical features that mimic the same featuresdisplayed on at least part of the surface of the cation-independent M6Preceptor binding face of IGF-II. Because a major contribution to thereceptor binding surface is the spatial arrangement of chemicallyinteractive moieties present within the sidechains of amino acids whichtogether define the receptor binding surface, a preferred embodiment ofthe present invention relates to designing and producing a syntheticorganic molecule having a framework that carries chemically interactivemoieties in a spatial relationship that mimics the spatial relationshipof the chemical moieties disposed on the amino acid sidechains whichconstitute the cation-independent M6P receptor binding face of IGF-II.Preferred chemical moieties, include but are not limited to, thechemical moieties defined by the amino acid side chains of amino acidsconstituting the cation-independent M6P receptor binding face of IGF-II.It is understood, therefore, that the receptor binding surface of theIGF-II analog need not comprise amino acid residues but the chemicalmoieties disposed thereon.

For example, upon identification of relevant chemical groups, theskilled artisan using a conventional computer program can design a smallmolecule having the receptor interactive chemical moieties disposed upona suitable carrier framework. Useful computer programs are described in,for example, Dixon (1992) Tibtech 10: 357-363; Tschinke et al. (1993) J.Med. Chem 36: 3863-3870; and Eisen el al. (1994) Proteins: Structure,Function, and Genetics 19: 199-221, the disclosures of which areincorporated herein by reference.

One particular computer program entitled “CAVEAT” searches a database,for example, the Cambridge Structural Database, for structures whichhave desired spatial orientations of chemical moieties (Bartlett et al.(1989) in “Molecular Recognition: Chemical and Biological Problems”(Roberts, S. M., ed) pp 182-196). The CAVEAT program has been used todesign analogs of tendamistat, a 74 residue inhibitor of α-amylase,based on the orientation of selected amino acid side chains in thethree-dimensional structure of tendamistat (Bartlett et al. (1989)supra).

Alternatively, upon identification of a series of analogs which mimicthe cation-independent M6P receptor binding activity of IGF-II, theskilled artisan may use a variety of computer programs which assist theskilled artisan to develop quantitative structure activity relationships(QSAR) and further to assist in the de novo design of additionalmorphogen analogs. Other useful computer programs are described in, forexample, Connolly-Martin (1991) Methods in Enzymology 203:587-613; Dixon(1992) supra; and Waszkowycz et al. (1994) J. Med. Chenm. 37: 3994-4002.

Targeting Moiety Affinities

Preferred targeting moieties bind to their target receptors with asubmicromolar dissociation constant. Generally speaking, lowerdissociation constants (e.g. less than 10⁻⁷ M, less than 10⁻⁸ M, or lessthan 10⁻⁹ M) are increasingly preferred. Determination of dissociationconstants is preferably determined by surface plasmon resonance asdescribed in Linnell et al. (2001) J. Biol. Chem. 276(26):23986-23991. Asoluble form of the extracellular domain of the target receptor (e.g.repeats 1-15 of the cation-independent M6P receptor) is generated andimmobilized to a chip through an avidin-biotin interaction. Thetargeting moiety is passed over the chip, and kinetic and equilibriumconstants are detected and calculated by measuring changes in massassociated with the chip surface.

Nucleic Acids and Expression Systems

Chimeric fusion proteins can be expressed in a variety of expressionsystems, including in vitro translation systems and intact cells. SinceM6P modification is not a prerequisite for targeting, a variety ofexpression systems including yeast, baculovirus and even prokaryoticsystems such as E. coli that do not glycosylate proteins are suitablefor expression of targeted therapeutic proteins. In fact, anunglycosylated protein generally has improved bioavailability, sinceglycosylated proteins are rapidly cleared from the circulation throughbinding to the mannose receptor in hepatic sinusoidal endothelium.

Alternatively, production of chimeric targeted lysosomal enzymes inmammalian cell expression system produces proteins with multiple bindingdeterminants for the cation-independent M6P receptor. Synergies betweentwo or more cation-independent M6P receptor ligands (e.g. M6P andIGF-II, or M6P and retinoic acid) can be exploited: multivalent ligandshave been demonstrated to enhance binding to the receptor by receptorcrosslinking.

In general, gene cassettes encoding the chimeric therapeutic protein canbe tailored for the particular expression system to incorporatenecessary sequences for optimal expression including promoters,ribosomal binding sites, introns, or alterations in coding sequence tooptimize codon usage. Because the protein is preferably secreted fromthe producing cell, a DNA encoding a signal peptide compatible with theexpression system can be substituted for the endogenous signal peptide.For example, for expression of β-glucuronidase and α-galactosidase Atagged with IGF-II in Leishmania, DNA cassettes encoding Leishmaniasignal peptides (GP63 or SAP) are inserted in place of the DNA encodingthe endogenous signal peptide to achieve optimal expression. Inmammalian expression systems the endogenous signal peptide may beemployed but if the IGF-II tag is fused at the 5′ end of the codingsequence, it could be desirable to use the IGF-II signal peptide.

CHO cells are a preferred mammalian host for the production oftherapeutic proteins. The classic method for achieving high yieldexpression from CHO cells is to use a CHO cell line deficient indihydrofolate reductase (DHFR), for example CHO line DUKX (O'Dell et al.(1998) Int. J. Biochem. Cell Biol, 30(7):767-71). This strain of CHOcells requires hypoxanthine and thymidine for growth. Co-transfection ofthe gene to be overexpressed with a DHFR gene cassette, on separateplasmids or on a single plasmid, permits selection for the DHFR gene andgenerally allows isolation of clones that also express the recombinantprotein of choice. For example, plasmid pcDNA3 uses the cytomegalovirus(CMV) early region regulatory region promoter to drive expression of agene of interest and pSV2DHFR to promote DHFR expression. Subsequentexposure of cells harboring the recombinant gene cassettes toincrementally increasing concentrations of the folate analogmethotrexate leads to amplification of both the gene copy number of theDHFR gene and of the co-transfected gene.

A preferred plasmid for eukaryotic expression in this system containsthe gene of interest placed downstream of a strong promoter such as CMV.An intron can be placed in the 3′ flank of the gene cassette. A DHFRcassette can be driven by a second promoter from the same plasmid orfrom a separate plasmid. Additionally, it can be useful to incorporateinto the plasmid an additional selectable marker such as neomycinphosphotransferase, which confers resistance to G418.

Another CHO expression system (Ulmasov et al. (2000) PNAS97(26):14212-14217) relies on amplification of the gene of interestusing G418 instead of the DHFR/methotrexate system described above. ApCXN vector with a slightly defective neomycin phosphotransferase drivenby a weak promoter (see, e.g., Niwa et al. (1991) Gene 108:193-200)permits selection for transfectants with a high copy number (>300) in asingle step.

Alternatively, recombinant protein can be produced in the human HEK 293cell line using expression systems based on the Epstein-Barr Virus (EBV)replication system. This consists of the EBV replication origin oriP andthe EBV ori binding protein, EBNA-1. Binding of EBNA-1 to oriP initiatesreplication and subsequent amplification of the extrachromosomalplasmid. This amplification in turn results in high levels of expressionof gene cassettes housed within the plasmid. Plasmids containing oriPcan be transfected into EBNA-1 transformed HEK 293 cells (commerciallyavailable from Invitrogen) or, alternatively, a plasmid such as pCEP4(commercially available from Invitrogen) which drives expression ofEBNA-1 and contains the EBV oriP can be employed.

In E. coli, the therapeutic proteins are preferably secreted into theperiplasmic space. This can be achieved by substituting for the DNAencoding the endogenous signal peptide of the LSD protein a nucleic acidcassette encoding a bacterial signal peptide such as the ompA signalsequence. Expression can be driven by any of a number of stronginducible promoters such as the lac, trp, or tac promoters. One suitablevector is pBAD/gIII (commercially available from Invitrogen) which usesthe Gene III signal peptide and the araBAD promoter.

In Vitro Refolding

One useful IGF-II targeting portion has three intramolecular disulfidebonds. GILT fusion proteins (for example GUS-GILT) in E. coli can beconstructed that direct the protein to the periplasmic space. IGF-II,when fused to the C-terminus of another protein, can be secreted in anactive form in the periplasm of E. coli (Wadensten et al. (1991)Biotechnol. Appl. Biochem. 13(3):412-21). To facilitate optimal foldingof the IGF-II moiety, appropriate concentrations of reduced and oxidizedglutathione are preferably added to the cellular milieu to promotedisulfide bond formation. In the event that a fusion protein withdisulfide bonds is incompletely soluble, any insoluble material ispreferably treated with a chaotropic agent such as urea to solubilizedenatured protein and refolded in a buffer having appropriateconcentrations of reduced and oxidized glutathione, or other oxidizingand reducing agents, to facilitate formation of appropriate disulfidebonds (Smith et al. (1989) J. Biol. Chem. 264(16):9314-21). For example,IGF-I has been refolded using 6M guanidine-HCl and 0.1 Mtris(2-carboxyethyl)phosphine reducing agent for denaturation andreduction of IGF-II (Yang et al. (1999) J. Biol. Chem.274(53):37598-604). Refolding of proteins was accomplished in 0.1MTris-HCl buffer (pH8.7) containing 1 mM oxidized glutathione, 10 mMreduced glutathione, 0.2M KCl and 1 mM EDTA.

Underglycosylation

Targeted therapeutic proteins are preferably underglycosylated: one ormore carbohydrate structures that would normally be present if theprotein were produced in a mammalian cell is preferably omitted,removed, modified, or masked, extending the half-life of the protein ina mammal. Underglycosylation can be achieved in many ways, several ofwhich are diagrammed in FIG. 1. As shown in FIG. 1, a protein may beactually underglycosylated, actually lacking one or more of thecarbohydrate structures, or functionally underglycosylated throughmodification or masking of one or more of the carbohydrate structures. Aprotein may be actually underglycosylated when synthesized, as discussedin Example 14, and may be completely unglycosylated (as when synthesizedin E. coli), partially unglycosylated (as when synthesized in amammalian system after disruption of one or more glycosylation sites bysite-directed mutagenesis), or may have a non-mammalian glycosylationpattern. Actual underglycosylation can also be achieved bydeglycosylation of a protein after synthesis. As discussed in Example14, deglycosylation can be through chemical or enzymatic treatments, andmay lead to complete deglycosylation or, if only a portion of thecarbohydrate structure is removed, partial deglycosylation.

In Vivo Expression

A nucleic acid encoding a therapeutic protein, preferably a secretedtherapeutic protein, can be advantageously provided directly to apatient suffering from a disease, or may be provided to a cell ex vivo,followed by administration of the living cell to the patient. In vivogene therapy methods known in the art include providing purified DNA(e.g. as in a plasmid), providing the DNA in a viral vector, orproviding the DNA in a liposome or other vesicle (see, for example, U.S.Pat. No. 5,827,703, disclosing lipid carriers for use in gene therapy,and U.S. Pat. No. 6,281,010, providing adenoviral vectors useful in genetherapy).

Methods for treating disease by implanting a cell that has been modifiedto express a recombinant protein are also well known. See, for example,U.S. Pat. No. 5,399,346, disclosing methods for introducing a nucleicacid into a primary human cell for introduction into a human. Althoughuse of human cells for ex vivo therapy is preferred in some embodiments,other cells such as bacterial cells may be implanted in a patient'svasculature, continuously releasing a therapeutic agent. See, forexample, U.S. Pat. Nos. 4,309,776 and 5,704,910.

Methods of the invention are particularly useful for targeting a proteindirectly to a subcellular compartment without requiring a purificationstep. In one embodiment, an IGF-II fusion protein is expressed in asymbiotic or attenuated parasitic organism that is administered to ahost. The expressed IGF-II fusion protein is secreted by the organism,taken up by host cells and targeted to their lysosomes.

In some embodiments of the invention, GILT proteins are delivered insitu via live Leishmania secreting the proteins into the lysosomes ofinfected macrophage. From this organelle, it leaves the cell and istaken up by adjacent cells not of the macrophage lineage. Thus, the GILTtag and the therapeutic agent necessarily remain intact while theprotein resides in the macrophage lysosome. Accordingly, when GILTproteins are expressed in situ, they are preferably modified to ensurecompatibility with the lysosomal environment. Human β-glucuronidase(human “GUS”), an exemplary therapeutic portion, normally undergoes aC-terminal peptide cleavage either in the lysosome or during transportto the lysosome (e.g. between residues 633 and 634 in GUS). Thus, inembodiments where a GUS-GILT construct is to be expressed by Leishmaniain a macrophage lysosome human GUS is preferably modified to render theprotein resistant to cleavage, or the residues following residue 633 arepreferably simply omitted from a GILT fusion protein. Similarly, IGF-II,an exemplary targeting portion, is preferably modified to increase itsresistance to proteolysis, or a minimal binding peptide (e.g. asidentified by phage display or yeast two hybrid) is substituted for thewildtype IGF-II moiety.

Administration

The targeted therapeutics produced according to the present inventioncan be administered to a mammalian host by any route. Thus, asappropriate, administration can be oral or parenteral, includingintravenous and intraperitoneal routes of administration. In addition,administration can be by periodic injections of a bolus of thetherapeutic or can be made more continuous by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an i.v. bag). In certain embodiments, the therapeutics of the instantinvention can be pharmaceutical-grade. That is, certain embodimentscomply with standards of purity and quality control required foradministration to humans. Veterinary applications are also within theintended meaning as used herein.

The formulations, both for veterinary and for human medical use, of thetherapeutics according to the present invention typically include suchtherapeutics in association with a pharmaceutically acceptable carriertherefor and optionally other ingredient(s). The carrier(s) can be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.Pharmaceutically acceptable carriers, in this regard, are intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is known in theart. Except insofar as any conventional media or agent is incompatiblewith the active compound, use thereof in the compositions iscontemplated. Supplementary active compounds (identified according tothe invention and/or known in the art) also can be incorporated into thecompositions. The formulations can conveniently be presented in dosageunit form and can be prepared by any of the methods well known in theart of pharmacy/microbiology. In general, some formulations are preparedby bringing the therapeutic into association with a liquid carrier or afinely divided solid carrier or both, and then, if necessary, shapingthe product into the desired formulation.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include oral or parenteral, e.g., intravenous,intradermal, inhalation, transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Ph can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

Useful solutions for oral or parenteral administration can be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990. Formulations for parenteral administration also caninclude glycocholate for buccal administration, methoxysalicylate forrectal administration, or cutric acid for vaginal administration. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Suppositories forrectal administration also can be prepared by mixing the drug with anon-irritating excipient such as cocoa butter, other glycerides, orother compositions that are solid at room temperature and liquid at bodytemperatures. Formulations also can include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration can include glycerol and other compositions of highviscosity. Other potentially useful parenteral carriers for thesetherapeutics include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration can contain as excipients, for example,lactose, or can be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oilysolutions for administration in the form of nasal drops, or as a gel tobe applied intranasally. Retention enemas also can be used for rectaldelivery.

Formulations of the present invention suitable for oral administrationcan be in the form of discrete units such as capsules, gelatin capsules,sachets, tablets, troches, or lozenges, each containing a predeterminedamount of the drug; in the form of a powder or granules; in the form ofa solution or a suspension in an aqueous liquid or non-aqueous liquid;or in the form of an oil-in-water emulsion or a water-in-oil emulsion.The therapeutic can also be administered in the form of a bolus,electuary or paste. A tablet can be made by compressing or moulding thedrug optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing, in a suitable machine, the drugin a free-flowing form such as a powder or granules, optionally mixed bya binder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered drug and suitable carrier moistened with an inert liquiddiluent.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients. Oral compositions preparedusing a fluid carrier for use as a mouthwash include the compound in thefluid carrier and are applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition can be sterile and can be fluid to the extentthat easy syringability exists. It can be stable under the conditions ofmanufacture and storage and can be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, and sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration can be in theform of a sterile aqueous preparation of the therapeutic which can be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems can also be used to present the therapeutic for bothintra-articular and ophthalmic administration.

Formulations suitable for topical administration, including eyetreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pasts; or solutions or suspensions such asdrops. Formulations for topical administration to the skin surface canbe prepared by dispersing the therapeutic with a dermatologicallyacceptable carrier such as a lotion, cream, ointment or soap. In someembodiments, useful are carriers capable of forming a film or layer overthe skin to localize application and inhibit removal. Where adhesion toa tissue surface is desired the composition can include the therapeuticdispersed in a fibrinogen-thrombin composition or other bioadhesive. Thetherapeutic then can be painted, sprayed or otherwise applied to thedesired tissue surface. For topical administration to internal tissuesurfaces, the agent can be dispersed in a liquid tissue adhesive orother substance known to enhance adsorption to a tissue surface. Forexample, hydroxypropylcellulose or fibrinogen/thrombin solutions can beused to advantage. Alternatively, tissue-coating solutions, such aspectin-containing formulations can be used.

For inhalation treatments, such as for asthma, inhalation of powder(self-propelling or spray formulations) dispensed with a spray can, anebulizer, or an atomizer can be used. Such formulations can be in theform of a finely comminuted powder for pulmonary administration from apowder inhalation device or self-propelling powder-dispensingformulations. In the case of self-propelling solution and sprayformulations, the effect can be achieved either by choice of a valvehaving the desired spray characteristics (i.e., being capable ofproducing a spray having the desired particle size) or by incorporatingthe active ingredient as a suspended powder in controlled particle size.For administration by inhalation, the therapeutics also can be deliveredin the form of an aerosol spray from a pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer. Nasal drops also can be used.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants generally are known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfilsidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the therapeutics typically are formulatedinto ointments, salves, gels, or creams as generally known in the art.

In one embodiment, the therapeutics are prepared with carriers that willprotect against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialsalso can be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. Microsomes and microparticles also can be used.

Oral or parenteral compositions can be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit formrefers to physically discrete units suited as unitary dosages for thesubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Generally, the therapeutics identified according to the invention can beformulated for parenteral or oral administration to humans or othermammals, for example, in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of the drug to targettissue for a time sufficient to induce the desired effect. Additionally,the therapeutics of the present invention can be administered alone orin combination with other molecules known to have a beneficial effect onthe particular disease or indication of interest. By way of exampleonly, useful cofactors include symptom-alleviating cofactors, includingantiseptics, antibiotics, antiviral and antifungal agents and analgesicsand anesthetics.

The effective concentration of the therapeutics identified according tothe invention that is to be delivered in a therapeutic composition willvary depending upon a number of factors, including the final desireddosage of the drug to be administered and the route of administration.The preferred dosage to be administered also is likely to depend on suchvariables as the type and extent of disease or indication to be treated,the overall health status of the particular patient, the relativebiological efficacy of the therapeutic delivered, the formulation of thetherapeutic, the presence and types of excipients in the formulation,and the route of administration. In some embodiments, the therapeuticsof this invention can be provided to an individual using typical doseunits deduced from the earlier-described mammalian studies usingnon-human primates and rodents. As described above, a dosage unit refersto a unitary, i.e. a single dose which is capable of being administeredto a patient, and which can be readily handled and packed, remaining asa physically and biologically stable unit dose comprising either thetherapeutic as such or a mixture of it with solid or liquidpharmaceutical diluents or carriers.

In certain embodiments, organisms are engineered to produce thetherapeutics identified according to the invention. These organisms canrelease the therapeutic for harvesting or can be introduced directly toa patient. In another series of embodiments, cells can be utilized toserve as a carrier of the therapeutics identified according to theinvention.

Therapeutics of the invention also include the “prodrug” derivatives.The term prodrug refers to a pharmacologically inactive (or partiallyinactive) derivative of a parent molecule that requiresbiotransformation, either spontaneous or enzymatic, within the organismto release or activate the active component. Prodrugs are variations orderivatives of the therapeutics of the invention which have groupscleavable under metabolic conditions. Prodrugs become the therapeuticsof the invention which are pharmaceutically active in vivo, when theyundergo solvolysis under physiological conditions or undergo enzymaticdegradation. Prodrug of this invention can be called single, double,triple, and so on, depending on the number of biotransformation stepsrequired to release or activate the active drug component within theorganism, and indicating the number of functionalities present in aprecursor-type form. Prodrug forms often offer advantages of solubility,tissue compatibility, or delayed release in the mammalian organism (see,Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp.352-401, Academic Press, San Diego, Calif, 1992). Moreover, the prodrugderivatives according to this invention can be combined with otherfeatures to enhance bioavailability.

EXAMPLES Example 1 GILT Constructs

IGF-II cassettes have been synthesized by ligation of a series ofoverlapping oligos and cloned into Pir1-SAT, a standard Leishmaniaexpression vector. 4 IGF-II cassettes have been made: one that encodesthe wildtype mature polypeptide, one with a Δ1-7 deletion, one with aY27L mutation, and one with both mutations. These mutations are reportedto reduce binding of IGF-II to the other receptors while not affectingbinding to the M6P receptor.

The coding sequence of human IGF-II is shown in FIGS. 2A-2B. The proteinis synthesized as a pre-pro-protein with a 24 amino acid signal peptideat the amino terminus and a 89 amino acid carboxy terminal region bothof which are removed post-translationally, reviewed in (O'Dell et al.(1998) Int. J. Biochem Cell Biol. 30(7):767-71. The mature protein is 67amino acids. A Leishmania codon optimized version of the mature IGF-IIis shown in FIG. 3 (Langford et al. (1992) Exp. Parasitol 74(3):360-1).This cassette was constructed by annealing overlapping oligonucleotideswhose sequences are shown in Table 3. Additional cassettes containing adeletion of amino acids 1-7 of the mature polypeptide (Δ1-7), alterationof residue 27 from tyrosine to leucine (Y27L) or both mutations(Δ1-7,Y27L) were made to produce IGF-II cassettes with specificity foronly the desired receptor as described below. To make the wildtypeIGF-II cassette, oligos GILT1-9 were annealed and ligated. To make theY27L cassette, oligos 1, 12, 3, 4, 5, 16, 7, 8 and 9 were annealed andligated. After ligation, the two cassettes were column purified.Wildtype and Y27L cassettes were amplified by PCR using oligos GILT 20and 10 and the appropriate template. To incorporate the Δ1-7 deletion,the two templates were amplified using oligos GILT 11 and 10. Theresulting 4 IGF-II cassettes (wildtype, Y27L, Δ1-7, and Y27LΔ1-7) werecolumn purified, digested with XbaI, gel purified and ligated to XbaIcut Pir1-SAT.

Gene cassettes were then cloned between the XmaI site (not shown)upstream of XbaI in the vector and the AscI site in such a way as topreserve the reading frame. An overlapping DAM methylase site at the 3′XbaI site permitted use of the 5′ XbaI site instead of the XmaI site forcloning. The AscI site adds a bridge of 3 amino acid residues.

TABLE 3 Oligonucleotides used in the construction of Pir-GILT vectors.SEQ NAME ID NO: SEQUENCE POSITION GILT 1  9GCGGCGGCGAGCTGGTGGACACGCTGCAGTT 48-97 top strand CGTGTGCGGCGACCGCGGCGILT 2 10 TTCTACTTCAGCCGCCCGGCCAGCCGCGTGA 98-147 top strandGCCGCCGCAGCCGCGGCAT GILT 3 11 CGTGGAGGAGTGCTGCTTCCGCAGCTGCGAC148-197 top strand CTGGCGCTGCTGGAGACGT GILT 4 12ACTGCGCGACGCCGGCGAAGTCGGAGTAAGA 198-237 top strand TCTAGAGCG GILT 5 13AGCGTGTCCACCAGCTCGCCGCCGCACAGCG 72-23 bottom TCTCGCTCGGGCGGTACGC GILT 614 GGCTGGCCGGGCGGCTGAAGTAGAAGCCGCG 122-73 bottom GTCGCCGCACACGAACTGCGILT 7 15 GCTGCGGAAGCAGCACTCCTCCACGATGCCG 172-123 bottomCGGCTGCGGCGGCTCACGC GILT 8 16 CTCCGACTTCGCCGGCGTCGCGCAGTACGTC223-173 bottom TCCAGCAGCGCCAGGTCGCA GILT 9 17CCGTCTAGAGCTCGGCGCGCCGGCGTACCGC 1-47 top strand CCGAGCGAGACGCTGT GILT 1018 CGCTCTAGATCTTACTCCGACTTCG 237-202 bottom GILT 11 19CCGTCTAGAGCTCGGCGCGCCGCTGTGCGGC 1-67, Δ23-43 top GGCGAGCTGGTGGAC GILT 1220 TTCCTGTTCAGCCGCCCGGCCAGCCGCGTGA 98-147 (Y27L) top GCCGCCGCAGCCGCGGCATGILT 16 21 GGCTGGCCGGGCGGCTGAACAGGAAGCCGCG 122-73 (Y27L) botGTCGCCGCACACGAACTGC GILT 20 22 CCGTCTAGAGCTCGGCGCGCCGGCG 1-25 top strand

The purpose of incorporating the indicated mutations into the IGF-IIcassette is to insure that the fusion proteins are targeted to theappropriate receptor. Human IGF-II has a high degree of sequence andstructural similarity to IGF-I (see, for example FIG. 7) and the B and Achains of insulin (Terasawa et al. (1994) Embo J. 13(23):5590-7).Consequently, it is not surprising that these hormones have overlappingreceptor binding specificities. IGF-II binds to the insulin receptor,the IGF-I receptor and the cation independent mannose 6-phosphate/IGF-IIreceptor (CIM6P/IGF-II). The CIM6P/IGF-II receptor is a dual activityreceptor acting as a receptor for IGF-II and as a mannose 6-phosphatereceptor involved in sorting of lysosomal hydrolases. For a number ofyears, these two activities were attributed to separate proteins untilit was determined that both activities resided in a single protein(Morgan et al. (1987) Nature 329(6137):301-7); (Tong et al (1988) J.Biol. Chem. 263(6):2585-8).

The most profound biological effects of IGF-II, such as its mitogeniceffect, are mediated through the IGF-I receptor rather than theCIM6P/IGF-II receptor, reviewed in (Ludwig et al (1995) Trends in CellBiology 5:202-206) also see (Korner et al. (1995) J. Biol. Chem.270(1):287-95). It is thought that the primary result of IGF-II bindingto the CIM6P/IGF-II receptor is transport to the lysosome for subsequentdegradation. This represents an important means of controlling IGF-IIlevels and explains why mice carrying null mutants of the CIM6P/IGF-IIreceptor exhibit perinatal lethality unless IGF-II is also deleted (Lauet al. (1994) Genes Dev. 8(24):2953-63); (Wang et al. (1994) Nature372(6505):464-7); (Ludwig et al (1996) Dev. Biol. 177(2):517-35). Inmethods of the present invention, it is desirable to have the fusionproteins bind to the CIM6P/IGF-II receptor. The Y27L and Δ1-7 mutationsreduce IGF-II binding to the IGF-I and insulin receptors withoutaltering the affinity for the CIM6P/IGF-II receptor (Sakano et al.(1991) J. Biol. Chem. 266(31):20626-35); (Hashimoto et al. (1995) J.Biol. Chem. 270(30):18013-8). Therefore, according to the invention,these mutant forms of IGF-II should provide a means of targeting fusionproteins specifically to the CIM6P/IGF-II receptor.

In one experiment, 4 different IGF-II cassettes with the appropriatesequences, wild type, Δ1-7, Y27L and Δ1-7/Y27L are made. β-GUS cassettesare fused to IGF-II cassettes and these constructs are put intoparasites. Alpha-galactosidase cassettes are also fused to the IGF-IIcassettes. GUS fusions have been tested and shown to produceenzymatically active protein.

One preferred construct, shown in FIGS. 4A-4E, includes the signalpeptide of the L. mexicana secreted acid phosphatase, SAP-1, cloned intothe XbaI site of a modified Pir1-SAT in which the single SalI site hasbeen removed. Fused in-frame is the mature β-GUS sequence, connected toan IGF-II tag by a bridge of three amino acids.

Example 2 GILT Protein Preparation

L. mexicana expressing and secreting β-GUS were grown at 26° C. in 100ml Standard Promastigote medium (M199 with 40 mM HEPES, pH 7.5, 0.1 mMadenine, 0.0005% hemin, 0.0001% biotin, 5% fetal bovine serum, 5%embryonic fluid, 50 units/ml penicillin, 50 μg/ml streptomycin and 50μg/ml nourseothricin). After reaching a density of approximately 5×10⁶promastigotes/ml, the promastigotes were collected by centrifugation for10 min. at 1000×g at room temperature; these promastigotes were used toinoculate 1 liter of low protein medium (M199 supplemented with 0.1 mMadenine, 0.0001% biotin, 50 units/ml penicillin and 50 μg/mlstreptomycin) at room temperature. The 1 liter cultures were containedin 2 liter capped flasks with a sterile stir bar so that the culturescould be incubated at 26° C. with gentle stirring. The 1 liter cultureswere aerated twice a day by moving them into a laminar flow hood,removing the caps and swirling vigorously before replacing the caps.When the cultures reached a density of 2-3×10⁷ promastigotes/ml, thecultures were centrifuged as described above except the promastigotepellet was discarded and the media decanted into sterile flasks. Theaddition of 434 g (NH₄)₂SO₄ per liter precipitated active GUS proteinfrom the medium; the salted out medium was stored at 4° C. overnight.Precipitated proteins were harvested either by centrifugation at10,500×g for 30 min. or filtration through Gelman Supor-800 membrane;the proteins were resuspended in 10 mM Tris pH 8, 1 mM CaCl₂ and storedat −80° C. until dialysis. The crude preparations from several liters ofmedium were thawed, pooled, placed in dialysis tubing (Spectra/Por-7,MWCO 25,000), and dialyzed overnight against two 1 liter volumes of DMEMwith bicarbonate (Dulbecco's Modified Eagle's Medium).

Example 3 GILT Uptake Assay

Skin fibroblast line GM4668 (human, NIGMS Human Genetic Mutant CellRepository) is derived from a patient with mucopolysaccharidosis VII;the cells therefore have little or no β-GUS activity. GM4668 cells aretherefore particularly useful for testing the uptake of GUS-GILTconstructs into human cells. GM4668 cells were cultured in 12-welltissue culture plates in Dulbecco's modified Eagle's medium (DMEM)supplemented with 15% (v/v) fetal calf serum at 37° C. in 5% CO₂.Fibroblasts were cultured overnight in the presence of about 150 unitsof preparations of Leishmania-expressed human β-glucuronidase (GUS),GUS-IGF-II fusion protein (GUS-GILT), or mutant GUS-IGF-II fusionprotein (GUSΔ-GILT) prepared as described in Example 2. Control wellscontained no added enzyme (DMEM media blank). After incubation, mediawas removed from the wells and assayed in triplicate for GUS activity.Wells were washed five times with 1 ml of 37° C. phosphate-bufferedsaline, then incubated for 15 minutes at room temperature in 0.2 ml oflysis buffer (10 mM Trig, pH7.5, 100 mM NaCl, 5 mM EDTA, 2 mM4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma),and 1% NP-40). Cell lysates were transferred to microfuge tubes, thenspun at 13,000 rpm for 5 minutes to remove cell debris. Three 10 μLaliquots of lysate were assayed for protein concentration (Pierce MicroBCA protein assay, Pierce, Ill.).

Three 38 μl aliquots of lysate were assayed for GUS activity using astandard fluorometric assay adapted from (Wolfe et al. (1996) Protocolsfor Gene Transfer in Neuroscience: Towards Gene Therapy of NeurologicalDisorders 263-274). Assays are done in disposable fluorimeter cuvettes.150 μl of reaction mix is added to each cuvette. 1 ml reaction mix is860 μl H2O, 100 μl 1M NaAcetate, 40 μl 25× β-GUS substrate mix. (25×β-GUS substrate mix is a suspension of 250 mg 4-methylumbelliferyl-β-Dglucuronide in 4.55 ml ethanol stored at −20° C. in a dessicator. 38 μlof sample are added to the reaction mix and the reaction is incubated at37° C. Reactions are terminated by addition of 2 ml stop solution (10.6g Na₂CO₃, 12.01 g glycine, H2O to 500 ml, pH 10.5). Fluorescence outputis then measured by fluorimeter.

Results of the uptake experiment indicate that the amount ofcell-associated GUS-GILT is 10-fold greater than that of the unmodifiedGUS (FIG. 5). The double mutant construct is about 5-fold more effectivethan unmodified GUS. These results indicate that the GILT technology isan effective means of targeting a lysosomal enzyme for uptake. Uptakecan also be verified using standard immunofluorescence techniques.

Example 4 Competition Experiments

To verify that the GILT—mediated uptake occurs via the IGF-II bindingsite on the cation-independent M6P receptor, competition experimentswere performed using recombinant IGF-II. The experimental design wasidentical to that described above except that GM4668 fibroblasts wereincubated with indicated proteins in DMEM minus serum +2% BSA for about18 hours. Each β-GUS derivative was added at 150 U per well. 2.85 μgIGF-II was added to each well for competition. This representsapproximately a 100 fold molar excess over GILT-GUS, a concentrationsufficient to compete for binding to the M6P/IGF-II receptor.

Results of the competition experiment are depicted in FIG. 6. In theabsence of IGF-II over 24 units of GILT-GUS/mg lysate were detected.Upon addition of IGF-II, the amount of cell associated GILT-GUS fell to5.4 U. This level is similar to the level of unmodified GUS taken up bythe fibroblasts. Thus, the bulk of the GILT protein uptake can becompeted by IGF-II indicating that the uptake is indeed occurringthrough a specific receptor-ligand interaction.

Example 5 Gene Product Expression in Serum Free Media

Expression products can also be isolated from serum free media. Ingeneral, the expression strain is grown in medium with serum, dilutedinto serum free medium, and allowed to grow for several generations,preferably 2-5 generations, before the expression product is isolated.For example, production of secreted targeted therapeutic proteins can beisolated from Leishmania mexicana promastigotes that are culturedinitially in 50 ml 1× M199 medium in a 75 cm2 flask at 27° C. When thecell density reaches 1-3×10⁷/ml, the culture is used to inoculate 1.2 Lof M199 media. When the density of this culture reaches about 5×106/ml,the cells are harvested by centrifugation, resuspended in 180 ml of thesupernatant and used to inoculate 12 L of “Zima” medium in a 16 Lspinner flask. The initial cell density of this culture is typicallyabout 5×10⁵/ml. This culture is expanded to a cell density of about1.0-1.7×10e⁷ cells/ml. When this cell density is reached, the cells areseparated from the culture medium by centrifugation and the supernatantis filtered at 4° C. through a 0.2μ filter to remove residualpromastigotes. The filtered media was concentrated from 12.0 L to 500 mlusing a tangential flow filtration device (MILLIPORE Prep/Scale-TFFcartridge).

Preferred growth media for this method are M199 and “Zima” growth media.However, other serum containing and serum free media are also useful.M199 growth media is as follows: (1 L batch)=200 ml 5× M199 (with phenolred pH indicator)+636 ml H₂O, 50.0 ml fetal bovine serum, 50.0 ml EFbovine embryonic fluid, 1.0 ml of 50 mg/ml nourseothricin, 2.0 ml of0.25% hemin in 50% triethanolamine, 10 ml of 10 mM adenine in 50 mMHepes pH 7.5, 40.0 ml of 1M Hepes pH 7.5, 1 ml of 0.1% biotin in 95%ethanol, 10.0 ml of penicillin/streptomycin. All sera used areinactivated by heat. The final volume=1 L and is filter sterilized.“Zima” modified M199 media is as follows: (20.0 L batch)=219.2 g M199powder (-)phenol red+7.0 g sodium bicarbonate, 200.0 ml of 10 mM adeninein 50 mM Hepes pH 7.5, 800.0 ml Of Hepes free acid pH 7.5, 20.0 ml 0.1%biotin in 95% ethanol, 200.0 ml penicillin/streptomycin, Finalvolume=20.0 L and is filter sterilized.

The targeted therapeutic proteins are preferably purified byConcanavalin A (ConA) chromatography. For example, when a culturereaches a density of >1.0×10⁷ promastigotes/ml, L. mexicana are removedby centrifugation, 10 min at 500×g. The harvested culture medium ispassed through a 0.2 μm filter to remove particulates before beingloaded directly onto a ConA-agarose column (4% cross-linked beadedagarose, Sigma). The ConA-agarose column is pretreated with 1 M NaCl, 20mM Tris pH 7.4, 5 mM each of CaCl₂, MgCl₂ and MnCl₂ and thenequilibrated with 5 volumes of column buffer (20 mM Tris pH 7.4, 1 mMCaCl₂, and 1 mM MnCl₂). A total of 179,800 units (nmol/hr) of GUSactivity (in 2 L) in culture medium is loaded onto a 22 ml ConA agarosecolumn. No activity is detectable in the flow through or wash. The GUSactivity is eluted with column buffer containing 200 mM methylmannopyranoside. Eluted fractions containing the activity peak arepooled and concentrated. Uptake and competition experiments wereperformed as described in Examples 3 and 4, except that the organismswere grown in serum-free medium and purified with ConA; about 350-600units of enzyme were applied to the fibroblasts. Results are shown inFIG. 8.

Example 6 Competition Experiments Using Denatured IGF-II as Competitor

The experiment in Example 4 is repeated using either normal or denaturedIGF-II as competitor. As in Example 4, the amount of cell-associatedGUS-GILT is reduced when coincubated with normal IGF-II concentrationsthat are effective for competition but, at comparable concentrations,denatured IGF-II has little or no effect.

Example 7 Enzyme Assays

Assays for GUS activity are performed as described in Example 3 and/oras described below.

Glass assay tubes are numbered in triplicate, and 100 μL of 2×GUSreaction mix are added to each tube. 2×GUS reaction mix is prepared byadding 100 mg of 4-methylumbelliferyl-β-D glucuronide to 14.2 mL 200 mMsodium acetate, pH adjusted to 4.8 with acetic acid. Up to 100 μL ofsample are added to each tube; water is added to a final reaction volumeof 200 μL. The reaction tubes are covered with parafilm and incubated ina 37° C. water bath for 1-2 hours. The reaction is stopped by additionof 1.8 mL of stop buffer (prepared by dissolving 10.6 g of Na₂CO₃ and12.01 g of glycine in a final volume of 500 mL of water, adjusting thepH to 10.5 and filter-sterilizing into a repeat-dispensor). Afluorimeter is then calibrated using 2 mL of stop solution as a blank,and the fluorescence is read from the remaining samples. A standardcurve is prepared using 1, 2, 5, 10, and 20 μL of a 166 μM4-methylumbelliferone standard in a final volume of 2 mL stop buffer.

The 4-methylumbelliferone standard solution is prepared by dissolving2.5 mg 4-methylumbelliferone in 1 mL ethanol and adding 99 mL of sterilewater, giving a concentration of approximately 200 nmol/mL. The preciseconcentration is determined spectrophotometrically. The extinctioncoefficient at 360 nm is 19,000 cm⁻¹ M⁻¹. For example, 100 μL is addedto 900 μL of stop buffer, and the absorbance at 360 nm is read. If thereading is 0.337, then the concentration of the standard solution is0.337×10 (dilution)/19,000=177 μM, which can then be diluted to 166 μMby addition of an appropriate amount of sterile water.

Example 8 Binding Uptake and Halflife Experiments

Binding of GUS-GILT proteins to the M6P/IGF-II receptor on fibroblastsare measured and the rate of uptake is assessed similar to publishedmethods (York et al. (1999) J. Biol. Chem. 274(2):1164-71). GM4668fibroblasts cultured in 12 well culture dishes as described above arewashed in ice-cold media minus serum containing 1% BSA. Ligand, (eitherGUS, GUS-GILT or GUS-ΔGILT, or control proteins) is added to cells incold media minus serum plus 1% BSA. Upon addition of ligand, the platesare incubated on ice for 30 minutes. After 30 minutes, ligand is removedand cells are washed quickly 5 times with ice cold media. Wells for the0 time point receive 1 ml ice cold stripping buffer (0.2 M Acetic acid,pH 3.5, 0.5M NaCl). The plate is then floated in a 37° water bath and0.5 ml prewarmed media is added to initiate uptake. At every stoppingpoint, 1 ml of stripping buffer is added. When the experiment is over,aliquots of the stripping buffer are saved for fluorometric assay ofβ-glucuronidase activity as described in Example 3. Cells are then lysedas described above and the lysate assayed for β-glucuronidase activity.Alternatively, immunological methods can be used to test the lysate forthe presence of the targeted therapeutic protein.

It is expected that GUS-GILT is rapidly taken up by fibroblasts in amatter of minutes once the temperature is shifted to 37° C. (York et al.(1999) J. Biol. Chem. 274(2):1164-71) and that the enzyme activitypersists in the cells for many hours.

Example 9 Protein Production in Mammalian Cells

CHO Cells

GUS-GILTΔ1-7 and GUSΔC18-GILTΔ1-7 were expressed in CHO cells using thesystem of Ulmasov et al. (2000) PNAS 97(26):14212-14217. Appropriategene cassettes were inserted into the Eco RI site of the pCXN vector,which was electroporated into CHO cells at 50 μF and 1,200 V in a 0.4-cmcuvette. Selection of colonies and amplification was mediated by 400μg/mL G418 for 2-3 weeks. The CHO cells were propagated in MEM mediasupplemented with 15% FBS, 1.2 mM glutamine, 50 μg/mL proline, and 1 mMpyruvate. For enzyme production cells were plated in multifloor flasksin MEM. Once cells reached confluence, collection medium (Weymouthmedium supplemented with 2% FBS, 1.2 mM glutamine, and 1 mM pyruvate)was applied to the cells. Medium containing the secreted recombinantenzyme was collected every 24-72 hours. A typical level of secretion forone GUS-GILTΔ1-7 cell line was 4000-5000 units/mL/24 hours.

A number of GUSΔC18-GILTΔ1-7 CHO lines were assayed for the amount ofsecreted enzyme produced. The six highest producers secreted between8600 and 14900 units/mL/24 hours. The highest producing line wasselected for collection of protein.

HEK 293 Cells

GUS-GILT cassettes were cloned into pCEP4 (Invitrogen) for expression inHEK 293 cells. Cassettes used included wild-type GUS-GILT; GUS-GILTΔ1-7;GUS-GILTY27L; GUSΔC18-GILTΔ1-7; GILTY27L, and GUS-GILTF19S/E12K.

HEK 293 cells were cultured to 50-80% confluency in 12-well platescontaining DMEM medium with 4 mM glutamine and 10% FBS. Cells weretransfected with pCEP-GUS-GILT DNA plasmids using FuGENE 6 (Roche) asdescribed by the manufacturer. 0.5 μg DNA and 2 μL of FuGENE 6 wereadded per well. Cells were removed from wells 2-3 days post-transfectionusing trypsin, then cultured in T25 cm² culture flasks containing theabove DMEM medium with 100 μg/mL hygromycin to select for a stablepopulation of transfected cells. Media containing hygromycin werechanged every 2-3 days. The cultures were expanded to T75 cm² cultureflasks within 1-2 weeks. For enzyme production cells were plated inmultifloor flasks in DMEM. Once cells reached confluence, collectionmedium (Weymouth medium supplemented with 2% FBS, 1.2 mM glutamine, and1 mM pyruvate) was applied to the cells. This medium has been optimizedfor CHO cells, not for 293 cells; accordingly, levels of secretion withthe HEK 293 lines may prove to be significantly higher in alternatemedia.

Levels of secreted enzyme are shown in Table 4.

TABLE 4 Cell line Recombinant Protein Units/mL/24 hours HEK293 2-1GUS-GILT 3151 HEK293 2-2 GUSΔC18-GILTΔ1-7 10367 HEK293 2-3 GUS-GILTΔ1-7186 HEK293 4-4 GILTY27L 3814 HEK293 3-5 GUS-GILTF19S/E12K 13223 HEK2933-6 GILTY27L 7948 CHO 15 GUSΔC18-GILTΔ1-7 18020

Example 10 Purification of GUS-GILT Fusion Proteins

Chromoatography, including conventional chromatography and affinitychromatography, can be used to purify GUS-GILT fusion proteins.

Conventional Chromatography

One procedure for purifying GUS-GILT fusion proteins produced inLeishmania is described in Example 2. An alternative procedure isdescribed in the following paragraph.

Culture supernatants from Leishmania mexicana cell lines expressingGUS-GILT fusions were harvested, centrifuged, and passed through a 0.2μfilter to remove cell debris. The supernatants were concentrated using atangential ultrafilter with a 100,000 molecular weight cutoff and storedat −80° C. Concentrated supernatants were loaded directly onto a columncontaining Concanavalin A (ConA) immobilized to beaded agarose. Thecolumn was washed with ConA column buffer (50 mM Tris pH 7.4, 1 mMCaCl₂, 1 mM MnCl₂) before mannosylated proteins including GUS-GILTfusions were eluted using a gradient of 0-0.2M methyl-α-D-pyranoside inthe ConA column buffer. Fractions containing glucuronidase activity(assayed as described in Example 7) were pooled, concentrated, and thebuffer exchanged to SP column buffer (25 mM sodium phosphate pH 6, 20 mMNaCl, 1 mM EDTA) in preparation for the next column. The concentratedfractions were loaded onto an SP fast flow column equilibrated in thesame buffer, and the column was washed with additional SP column buffer.The GUS-GILT fusions were eluted from the column in two steps: 1) agradient of 0-0.15 M glucuronic acid in 25 mM sodium phosphate pH 6 and10% glycerol, followed by 0.2 M glucuronic acid, 25 mM sodium phosphatepH 6, 10% glycerol. Fractions containing glucuronidase activity werepooled, and the buffer exchanged to 20 mM potassium phosphate pH 7.4.These pooled fractions were loaded onto an HA-ultrogel columnequilibrated with the same buffer. The GUS-GILT fusion proteins wereeluted with an increasing gradient of phosphate buffer, from 145-340 mMpotassium phosphate pH 7.4. The fractions containing glucuronidaseactivity were pooled, concentrated, and stored at −80° C. in 20 mM TrispH 8 with 25% glycerol.

A conventional chromatography method for purifying GUS-GILT fusionproteins produced in mammalian cells is described in the followingparagraphs.

Mammalian cells overexpressing a GUS-GILT fusion protein are grown toconfluency in Nunc Triple Flasks, then fed with serum-free medium(Waymouth MB 752/1) supplemented with 2% fetal bovine serum to collectenzyme for purification. The medium is harvested and the flasks arerefed at 24 hour intervals. Medium from several flasks is pooled andcentrifuged at 5000×g for 20 minutes at 4° C. to remove detached cells,etc. The supernatant is removed and aliquots are taken for a β-GUSassay. The medium can now be used directly for purification or frozen at−20° C. for later use.

1 L of secretion medium is thawed at 37° C. (if frozen), filteredthrough a 0.2μ filter, and transferred to a 4 L beaker. The volume ofthe medium is diluted 4-fold by addition of 3 L of dd water to reducethe salt concentration; the pH of the diluted medium is adjusted to 9.0using 1 M Tris base. 50 mL of DEAE-Sephacel pre-equilibrated with 10 mMTris pH 9.0 is added to the diluted medium and stirred slowly with alarge stirring bar at 4° C. for 2 hours. (A small aliquot can beremoved, microfuged, and the supernatant assayed to monitor binding.)When binding is complete, the resin is collected on a fritted glassfunnel and washed with 750 mL of 10 mM Tris pH 9.0 in several batches.The resin is transferred to a 2.5 cm column and washed with anadditional 750 mL of the same buffer at a flow rate of 120 mL/hour. TheDEAE column is eluted with a linear gradient of 0-0.4 M NaCl in 10 mMTris pH 9.0. The fractions containing the GUS-GILT fusion proteins aredetected by 4-methylumbelliferyl-β-D glucuronide assay, pooled, andloaded onto a 600 mL column of Sephacryl S-200 equilibrated with 25 mMTris pH 8, 1 mM β-glycerol phosphate, 0.15 M sodium chloride and elutedwith the same buffer.

The fractions containing the GUS-GILT fusion proteins are pooled anddialyzed with 3×4 L of 25 mM sodium acetate pH 5.5, 1 mM β-glycerolphosphate, 0.025% sodium azide. The dialyzed enzyme is loaded at a flowrate of 36 mL/hour onto a 15 mL column of CM-Sepharose equilibrated with25 mM sodium acetate pH 5.5, 1 mM β-glycerol phosphate, 0.025% sodiumazide. It is then washed with 10 column volumes of this same buffer. TheCM column is eluted with a linear gradient of 0-0.3 M sodium chloride inthe equilibration buffer. The fractions containing the GUS-GILT fusionproteins are pooled and loaded onto a 2.4×70 cm (Bed volume=317 mL)column of Sephacryl S-300 equilibrated with 10 mM Tris pH 7.5, 1 mMβ-glycerol phosphate, 0.15 M NaCl at a flow rate of 48 mL/hour. Thefractions containing the fusion proteins are pooled; the pool is assayedfor GUS activity and for protein concentration to determine specificactivity. Aliquots are run on SDS-PAGE followed by Coomassie or silverstaining to confirm purity. If a higher concentration of enzyme isrequired, Amicon Ultrafiltration Units with an XM-50 membrane (50,000molecular weight cutoff) or Centricon C-30 units (30,000 molecularweight cutoff) can be used to concentrate the fusion protein. The fusionprotein is stored at −80° C. in the 10 mM Tris pH 7.5, 1 mM sodiumβ-glycerol phosphate, 0.15 M NaCl buffer.

Affinity Chromatography

Affinity chromatography conditions are essentially as described in Islamet al. (1993) J. Biol. Chem. 268(30):22627-22633. Conditioned mediumfrom mammalian cells overexpressing a GUS-GILT fusion protein (collectedand centrifuged as described above for conventional chromatography) isfiltered through a 0.22μ filter. Sodium chloride (crystalline) is addedto a final concentration of 0.5M, and sodium azide is added to a finalconcentration of 0.025% by adding 1/400 volume of a 10% stock solution.The medium is applied to a 5 mL column of anti-humanβ-glucuronidase-Affigel 10 (pre-equilibrated with Antibody SepharoseWash Buffer: 10 mM Tris pH 7.5, 10 mM potassium phosphate, 0.5 M NaCl,0.025% sodium azide) at a rate of 25 mL/hour at 4° C. Fractions arecollected and monitored for any GUS activity in the flow-through. Thecolumn is washed at 36 mL/hour with 10-20 column volumes of AntibodySepharose Wash Buffer. Fractions are collected and monitored for GUSactivity. The column is eluted at 36 mL/hour with 50 mL of 10 mM sodiumphosphate pH 5.0+3.5 M MgCl₂. 4 mL fractions are collected and assayedfor GUS activity. Fractions containing the fusion protein are pooled,diluted with an equal volume of P6 buffer (25 mM Tris pH 7.5, 1 mMβ-glycerol phosphate, 0.15 mM NaCl, 0.025% sodium azide) and desaltedover a BioGel P6 column (pre-equilibrated with P6 buffer) to remove theMgCl₂ and to change the buffer to P6 buffer for storage. The fusionprotein is eluted with P6 buffer, fractions containing GUS activity arepooled, and the pooled fractions assayed for GUS activity and forprotein. An SDS-PAGE gel stained with Coomassie Blue or silver stain isused to confirm purity. The fusion protein is stored frozen at −80° C.in P6 buffer for long-term stability.

Example 11 Uptake Experiments on Mammalian-produced Proteins

Culture supernatants from HEK293 cell lines or CHO cell lines producingGUS or GUS-GILT constructs were harvested through a 0.2 μm filter toremove cells GM 4668 fibroblasts were cultured in 12-well tissue cultureplates in DMEM supplemented with 15% (v/v) fetal calf serum at 37° C. in5% CO₂. Cells were washed once with uptake medium (DMEM+2% BSA (SigmaA-7030)) at 37° C. Fibroblasts were then cultured (3-21 hours) with1000-4000 units of enzyme per mL of uptake medium. In some experiments,competitors for uptake were added. Mannose-6-phosphate (Calbiochem444100) was added to some media at concentrations from 2-8 mM and purerecombinant IGF-II (Cell Sciences OU100) was added to some media at 2.86mM, representing a 10-100 fold molar excess depending on the quantity ofinput enzyme. Uptake was typically measured in triplicate wells.

After incubation, the media were removed from the wells and assayed induplicate for GUS activity. Wells were washed five times with 1 mL of37° C. phosphate-buffered saline, then incubated for 15 minutes at roomtemperature in 0.2 mL of lysis buffer (10 mM Tris, pH 7.5, 100 mM NaCl,5 mM EDTA, and 1% NP-40). Cell lysates were transferred to microfugetubes and spun at 13,000 rpm for 5 minutes to remove cell debris. Two 10μL aliquots of lysate were assayed for GUS activity using a standardfluorometric assay. Three 10 μL aliquots of lysate were assayed forprotein concentration (Pierce Micro BCA protein assay, Pierce, Ill.).

An initial experiment compared uptake of CHO-produced GUS-GILTΔ1-7 withCHO-produced GUSΔC18-GILTβ1-7. As shown in Table 5, the GUSΔC18-GILTΔ1-7protein, which was engineered to eliminate a potential protease cleavagesite, has significantly higher levels of uptake levels that can beinhibited by IGF-II and by M6P. In contrast, the uptake of a recombinantGUS produced in mammalian cells lacking the IGF-II tag was unaffected bythe presence of excess IGF-II but was completely abolished by excessM6P. In this experiment, uptake was performed for 18 hours.

TABLE 5 Input Uptake +IGF-II % IGF-II +M6P % M6P Enzyme units (units/mg)(units/mg) inhibition (units/mg) inhibition CHO GUS-GILTΔ1-7 982 310 ±27  84 ± 20 73 223 ± 36 28 CHO GUSΔC18- 1045  704 ± 226 258 ± 50 63 412± 79 41 GILTΔ1-7 CHO GUS 732 352 ± 30 336 ± 77 5   1 ± 0.2 99.7

A subsequent experiment assessed the uptake of CHO- and HEK293-producedenzymes by human fibroblasts from MPSVII patients. In this experiment,uptake was for 21 hours.

TABLE 6 +IGF-II % IGF-II Enzyme Input units Uptake (units/mg) Uptake(units/mg) inhibition CHO GUSΔC18-GILTΔ1-7 2812  4081 ± 1037 1007 ± 13275 HEK GUS-GILT 2116 1432 ± 196 HEK GUSΔC18-GILTΔ1-7 3021 5192 ± 3201207 ± 128 77 HEK GUS-GILTY27L 3512 1514 ± 203 HEK GUS-GILTF19SE12K 32114227 ± 371 388 ± 96 90.8 HEK GUS-GILTF19S 3169 4733 ± 393 439 ± 60 90.7

A further experiment assessed the uptake of selected enzymes in thepresence of IGF-II, 8 mM M6P, or both inhibitors. Uptake was measuredfor a period of 22.5 hours.

TABLE 7 +IGF-II + Uptake +IGF-II % IGF- +M6P M6P % IGF- Input (units/(units/ II (units/ % M6P (units/ II + M6P Enzyme units mg) mg)inhibition mg) inhibition mg) inhibition CHO 1023 1580 ± 150 473 ± 27 70639 ± 61 60 0 ± 1 100 GUSΔC18- GILTΔ1-7 HEK GUS- 880 1227 ± 76  22 ± 298.2 846 ± 61 31 0 ± 3 100 GILTF19S E12K HEK GUS- 912 1594 ± 236 217 ±17 86 952 ± 96 60 15 ± 2  99.06 GILTF19S

The experiments described above show that CHO and HEK293 productionsystems are essentially equivalent in their ability to secretefunctional recombinant proteins. The experiments also show that thepresence of excess IGF-II diminishes uptake of tagged proteins by70-90+%, but does not markedly affect uptake of untagged protein (4.5%),indicating specific IGF-II-mediated uptake of the mammalian-producedprotein. Unlike Leishmania-produced proteins, the enzymes produced inmammalian cells are expected to contain M6P. The presence of two ligandson these proteins capable of directing uptake through the M6P/IGF-IIreceptor implies that neither excess IGF-II nor excess M6P shouldcompletely abolish uptake. Furthermore, since the two ligands bind todiscrete locations on the receptor, binding to the receptor via oneligand should not be markedly affected by the presence of an excess ofthe other competitor.

Example 12 In Vivo Therapy

Initially, GUS minus mice can be used to assess the effectiveness ofGUS-GILT and derivatives thereof in enzyme replacement therapy. GUSminus mice are generated by heterozygous matings ofB6.C-H-2^(bml)/ByBIR-gus^(mps)/+mice as described by Birkenmeier et al.(1989) J. Clin. Invest 83(4):1258-6. Preferably, the mice are tolerantto human β-GUS. The mice may carry a transgene with a defective copy ofhuman β-GUS to induce immunotolerance to the human protein (Sly et al.(2001) PNAS 98:2205-2210). Alternatively, human β-GUS (e.g. as aGUS-GILT protein) can be administered to newborn mice to induceimmunotolerance. However, because the blood-brain barrier is not formeduntil about day 15 in mice, it is simpler to determine whether GILT-GUScrosses the blood-brain barrier when initiating injections in mice olderthan 15 days; transgenic mice are therefore preferable.

The initial experiment is to determine the tissue distribution of thetargeted therapeutic protein. At least three mice receive a CHO-producedGILT-tagged β-GUS protein referred to herein as GUSΔC18-GILTΔ1-7, inwhich GUSΔ18, a β-GUS protein omitting the last eighteen amino acids ofthe protein, is fused to the N-terminus of Δ1-7 GILT, an IGF-II proteinmissing the first seven amino acids of the mature protein. Other micereceive either β-GUS, a buffer control, or a GUSΔC18-GILTΔ1-7 proteintreated with periodate and sodium borohydride as described in Example14. Generally, preferred doses are in the range of 0.5-7 mg/kg bodyweight. In one example, the enzyme dose is 1 mg/kg body weightadministered intravenously, and the enzyme concentration is about 1-3mg/mL. In addition, at least three mice receive a dose of 5 mg/kg bodyweight of GUSΔC18-GILTΔ1-7 protein treated with periodate and sodiumborohydride. After 24 hours, the mice are sacrificed and the followingorgans and tissues are isolated: liver, spleen, kidney, brain, lung,muscle, heart, bone, and blood. Portions of each tissue are homogenizedand the β-GUS enzyme activity per mg protein is determined as describedin Sly et al. (2001) PNAS 98:2205-2210. Portions of the tissues areprepared for histochemistry and/or histopathology carried out bypublished methods (see, e.g., Vogler et al. (1990) Am J. Pathol.136:207-217).

Further experiments include multiple injection protocols in which themice receive weekly injections at a dose of 1 mg/kg body weight. Inaddition, measurement of the half-life of the periodate-modified enzymeis determined in comparison with untreated enzyme as described inExample 14.

Two other assay formats can be used. In one format, 3-4 animals aregiven a single injection of 20,000 U of enzyme in 100 μl enzyme dilutionbuffer (150 mM NaCl, 10 mM Tris, pH7.5). Mice are killed 72-96 hourslater to assess the efficacy of the therapy. In a second format, miceare given weekly injections of 20,000 units over 3-4 weeks and arekilled 1 week after the final injection. Histochemical andhistopathologic analysis of liver, spleen and brain are carried out bypublished methods (Birkenmeier et al. (1991) Blood 78(10:3081-92; Sandset al. (1994) J. Clin. Invest 93(6):2324-31; Daly et al. (1999) Proc.Natl. Acad. Sci. USA 96(5):2296-300). In the absence of therapy, cells(e.g. macrophages and Kupffer cells) of GUS minus mice develop largeintracellular storage compartments resulting from the buildup of wasteproducts in the lysosomes. It is anticipated that in cells in micetreated with GUS-GILT constructs, the size of these compartments will bevisibly reduced or the compartments will shrink until they are no longervisible with a light microscope.

Similarly, humans with lysosomal storage diseases will be treated usingconstructs targeting an appropriate therapeutic portion to theirlysosomes. In some instances, treatment will take the form of regular(e.g. weekly) injections of a GILT protein. In other instances,treatment will be achieved through administration of a nucleic acid topermit persistent in vivo expression of a GILT protein, or throughadministration of a cell (e.g. a human cell, or a unicellular organism)expressing the GILT protein in the patient. For example, the GILTprotein can be expressed in situ using a Leishmania vector as describedin U.S. Pat. No. 6,020,144, issued Feb. 1, 2000; U.S. ProvisionalApplication No. 60/250,446; and U.S. Provisional Application No.60/290,281, “Protozoan Expression Systems for Lysosomal Storage DiseaseGenes”, filed May 11, 2001.

Targeted therapeutic proteins of the invention can also be administered,and their effects monitored, using methods (enzyme assays, histochemicalassays, neurological assays, survival assays, reproduction assays, etc.)previously described for use with GUS. See, for example, Vogler et al.(1993) Pediatric Res. 34(6):837-840; Sands et al. (1994) J. Clin.Invest. 93:2324-2331; Sands et al. (1997) J. Clin. Invest. 99:1596-1605;O'Connor et al. (1998) J. Clin. Invest, 101:1394-1400; and Soper et al.(1999) 45(2):180-186.

Example 13

The objective of these experiments is to evaluate the efficacy ofGILT-modified alpha-galactosidase A (α-GAL A) as an enzyme replacementtherapy for Fabry's disease.

Fabry's disease is a lysosomal storage disease resulting frominsufficient activity of α-GAL A, the enzyme responsible for removingthe terminal galactose from GL-3 and other neutral sphingolipids. Thediminished enzymatic activity occurs due to a variety of missense andnonsense mutations in the x-linked gene. Accumulation of GL-3 is mostprevalent in lysosomes of vascular endothelial cells of the heart,liver, kidneys, skin and brain but also occurs in other cells andtissues. GL-3 buildup in the vascular endothelial cells ultimately leadsto heart disease and kidney failure.

Enzyme replacement therapy is an effective treatment for Fabry'sdisease, and its success depends on the ability of the therapeuticenzyme to be taken up by the lysosomes of cells in which GL-3accumulates. The Genzyme product, Fabrazyme, is recombinant α-GAL Aproduced in DUKX B11 CHO cells that has been approved for treatment ofFabry's patients in Europe due to its demonstrated efficacy.

The ability of Fabrazyme to be taken up by cells and transported to thelysosome is due to the presence of mannose 6-phosphate (M6P) on itsN-linked carbohydrate. Fabrazyme is delivered to lysosomes throughbinding to the mannose-6-phosphate/IGF-II receptor (M6P/IGF-Iir),present on the cell surface of most cell types, and subsequent receptormediated endocytosis. Fabrazyme reportedly has three N-linkedglycosylation sites at ASN residues 108, 161, and 184. The predominantcarbohydrates at these positions are fucosylated biantennarybisialylated complex, monophosphorylated mannose-7 oligomannose, andbiphosphorylated mannose-7 oligomannose, respectively.

The glycosylation independent lysosomal targeting (GILT) technology ofthe present invention directly targets therapeutic proteins to thelysosome via a different interaction with the M6P/IGF-Iir. A targetingligand is derived from mature human IGF-II, which also binds with highaffinity to the M6P/IGF-Iir. In current applications, the IGF-II tag isprovided as a c-terminal fusion to the therapeutic protein, althoughother configurations are feasible including cross-linking. Thecompetency of GILT-modified enzymes for uptake into cells has beenestablished using GILT-modified β-glucuronidase, which is efficientlytaken up by fibroblasts in a process that is competed with excessIGF-II. Advantages of the GILT modification are increased binding to theM6P/IGF-II receptor, enhanced uptake into lysosomes of target cells,altered or improved pharmacokinetics, and expanded, altered or improvedrange of tissue distribution. The improved range of tissue distributionscould include delivery of GILT-modified α-GAL A across the blood-brainbarrier since IGF proteins demonstrably cross the blood-brain barrier.

Another advantage of the GILT system is the ability to produceuptake-competent proteins in non-mammalian expression systems where M6Pmodifications do not occur. In certain embodiments, GILT-modifiedprotein will be produced primarily in CHO cells. In certain others, theGILT tag will be placed at the c-terminus of α-GAL A although theinvention is not so limited.

Example 14 Underglycosylated Therapeutic Proteins

The efficacy of a targeted therapeutic can be increased by extending theserum half-life of the targeted therapeutic. Hepatic mannose receptorsand asialoglycoprotein receptors eliminate glycoproteins from thecirculation by recognizing specific carbohydrate structures (Lee et al.(2002) Science 295(5561):1898-1901; Ishibashi et al. (1994) J. Biol.Chem. 269(45):27803-6). In some embodiments, the present inventionpermits targeting of a therapeutic to lysosomes and/or across the bloodbrain barrier in a manner dependent not on a carbohydrate, but on apolypeptide or an analog thereof. Actual underglycosylation of theseproteins is expected to greatly increase their half-life in thecirculation, by minimizing their removal from the circulation by themannose and asialoglycoprotein receptors. Similarly, functionaldeglycosylation (e.g. by modifying the carbohydrate residues on thetherapeutic protein, as by periodate/sodium borohydride treatment)achieves similar effects by interfering with recognition of thecarbohydrate by one or more clearance pathways. Nevertheless, becausetargeting of the protein relies, in most embodiments, onprotein-receptor interactions rather than carbohydrate-receptorinteractions, modification or elimination of glycosylation should notadversely affect targeting of the protein to the lysosome and/or acrossthe blood brain barrier.

Any lysosomal enzyme using a peptide targeting signal such as IGF-II canbe chemically or enzymatically deglycosylated or modified to produce atherapeutic with the desirable properties of specific lysosomaltargeting plus long serum half-life. In the case of some lysosomalstorage diseases where it might be important to deliver the therapeuticto macrophage or related cell types via mannose receptor, fullyglycosylated therapeutics can be used in combination withunderglycosylate targeted therapeutics to achieve targeting to thebroadest variety of cell types.

Proteins Underglycosylated when Synthesized

In some cases it will be preferable to produce the targeted therapeuticprotein initially in a system that does not produce a fully glycosylatedprotein. For example, a targeted therapeutic protein can be produced inE. coli, thereby generating a completely unglycosylated protein.Alternatively, an unglycosylated protein is produced in mammalian cellstreated with tunicamycin, an inhibitor of Dol-PP-GlcNAc formation. If,however, a particular targeted therapeutic does not fold correctly inthe absence of glycosylation, it is preferably produced initially as aglycosylated protein, and subsequently deglycosylated or renderedfunctionally underglycosylated.

Underglycosylated targeted therapeutic proteins can also by prepared byengineering a gene encoding the targeted therapeutic protein so that anamino acid that normally serves as an acceptor for glycosylation ischanged to a different amino acid. For example, an asparagine residuethat serves as an acceptor for N-linked glycosylation can be changed toa glutamine residue, or another residue that is not a glycosylationacceptor. This conservative change is most likely to have a minimalimpact on enzyme structure while eliminating glycosylation at the site.Alternatively, other amino acids in the vicinity of the glycosylationacceptor can be modified, disrupting a recognition motif forglycosylation enzymes without necessarily changing the amino acid thatwould normally be glycosylated.

In the case of GUS, removal of any one of 4 potential glycosylationsites lessens the amount of glycosylation while retaining ample enzymeactivity (Shipley et al. (1993) J. Biol. Chem. 268(16):12193-8). Removalof some sets of two glycosylation sites from GUS still permitssignificant enzyme activity. Removal of all four glycosylation siteseliminates enzyme activity, as does treatment of cells with tunicamycin,but deglycosylation of purified enzyme results in enzymatically activematerial. Therefore, loss of activity associated with removal of theglycosylation sites is likely due to incorrect folding of the enzyme.

Other enzymes, however, fold correctly even in the absence ofglycosylation. For example, bacterial β-glucuronidase is naturallyunglycosylated, and can be targeted to a mammalian lysosome and/oracross the blood brain barrier using the targeting moieties of thepresent invention. Such enzymes can be synthesized in an unglycosylatedstate, rather than, for example, synthesizing them as glycosylatedproteins and subsequently deglycosylating them.

Deglycosylation

If the targeted therapeutic is produced in a mammalian cell culturesystem, it is preferably secreted into the growth medium, which can beharvested, permitting subsequent purification of the targetedtherapeutic by, for example, chromatographic purification protocols,such as those involving ion exchange, gel filtration, hydrophobicchromatography, ConA chromatography, affinity chromatography orimmunoaffinity chromatography.

Chemical deglycosylation of glycoproteins can be achieved in a number ofways, including treatment with trifluoromethane sulfonic acid (TFMS), ortreatment with hydrogen fluoride (HF).

Chemical deglycosylation by TFMS (Sojar et al. (1989) J. Biol. Chem.264(5):2552-9; Sojar et al. (1987) Methods Enzymol. 138:341-50): 1 mgGILT-GUS is dried under vacuum overnight. The dried protein is treatedwith 150 μl TFMS at 0° C. for 0.5-2 hours under nitrogen with occasionalshaking. The reaction mix is cooled to below −20° C. in a dryice-ethanol bath and the reaction is neutralized by the gradual additionof a prechilled (−20° C.) solution of 60% pyridine in water. Theneutralized reaction mix is then dialyzed at 4° C. against severalchanges of NH₄HCO₃ at pH 7.0. Chemical deglycosylation with TFMS canresult in modifications to the treated protein including methylation,succinimide formation and isomerization of aspartate residues (Douglasset al. (2001) J. Protein Chem. 20(7):571-6).

Chemical deglycosylation by HF (Sojar et al. (1987) Methods Enzymol.138:341-50): The reaction is carried out in a closed reaction systemsuch as can be obtained from Peninsula Laboratories, Inc. 10 mg GILT-GUSis vacuum dried and placed in a reaction vessel which is then connectedto the HF apparatus. After the entire HF line is evacuated, 10 mLanhydrous HF is distilled over from the reservoir with stirring of thereaction vessel. The reaction is continued for 1-2 hours at 0° C.Afterwards, a water aspirator removes the HF over 15-30 minutes.Remaining traces of HF are removed under high vacuum. The reactionmixture is dissolved in 2 mL 0.2M NaOH to neutralize any remaining HFand the pH is readjusted to 7.5 with cold 0.2M HCl.

Enzymatic deglycosylation (Thotakura et al. (1987) Methods Enzymol.138:350-9): N-linked carbohydrates can be removed completely fromglycoproteins using protein N-glycosidase (PNGase) A or F. In oneembodiment, a glycoprotein is denatured prior to treatment with aglycosidase to facilitate action of the enzyme on the glycoprotein; theglycoprotein is subsequently refolded as discussed in the “In vitrorefolding” section above. In another embodiment, excess glycosidase isused to treat a native glycoprotein to promote effectivedeglycosylation.

In the case of a targeted therapeutic protein that is actuallyunderglycosylated, it is possible that the reduced glycosylation willreveal protease-sensitive sites on the targeted therapeutic protein,which will diminish the half-life of the protein. N-linked glycosylationis known to protect a subset of lysosomal enzymes from proteolysis(Kundra at al. (1999) J. Biol. Chem. 274(43):31039-46). Suchprotease-sensitive sites are preferably engineered out of the protein(e.g. by site-directed mutagenesis). As discussed below, the risk ofrevealing either a protease-sensitive site or a potential epitope can beminimized by incomplete deglycosylation or by modifying the carbohydratestructure rather than omitting the carbohydrate altogether.

Modification of Carbohydrate Structure or Partial Deglycosylation

In some embodiments, the therapeutic protein is partiallydeglycosylated. For example, the therapeutic protein can be treated withan endoglycosidase such as endoglycosidase H, which cleaves N-linkedhigh mannose carbohydrate but not complex type carbohydrate leaving asingle GlcNAc residue linked to the asparagine. A therapeutic proteintreated in this way will lack high mannose carbohydrate, reducinginteraction with the hepatic mannose receptor. Even though this receptorrecognizes terminal GlcNAc, the probability of a productive interactionwith the single GlcNAc on the protein surface is not as great as with anintact high mannose structure. If the therapeutic protein is produced inmammalian cells, any complex carbohydrate present on the protein willremains unaffected by the endoH treatment and may be terminallysialylated, thereby diminishing interactions with hepatic carbohydraterecognizing receptors. Such a protein is therefore likely to haveincreased half-life. At the same time, steric hinderance by theremaining carbohydrate should shield potential epitopes on the proteinsurface from the immune system and diminish access of proteases to theprotein surface (e.g. in the protease-rich lysosomal environment).

In other embodiments, glycosylation of a therapeutic protein ismodified, e.g. by oxidation, reduction, dehydration, substitution,esterification, alkylation, sialylation, carbon-carbon bond cleavage, orthe like, to reduce clearance of the therapeutic protein from the blood.In some preferred embodiments, the therapeutic protein is notsialylated. For example, treatment with periodate and sodium borohydrideis effective to modify the carbohydrate structure of most glycoproteins.Periodate treatment oxidizes vicinal dials, cleaving the carbon-carbonbond and replacing the hydroxyl groups with aldehyde groups; borohydridereduces the aldehydes to hydroxyls. Many sugar residues include vicinaldiols and, therefore, are cleaved by this treatment. As shown in FIG.9A, a protein may be glycosylated on an asparagine residue with a highmannose carbohydrate that includes N-acetylglucosamine residues near theasparagine and mannose residues elsewhere in the structure. As shown inFIG. 9B, the terminal mannose residues have three consecutive carbonswith hydroxyl groups; both of the carbon-carbon bonds involved arecleaved by periodate treatment. Some nonterminal mannose residues alsoinclude a vicinal diol, which would similarly be cleaved. Nevertheless,while this treatment converts cyclic carbohydrates into linearcarbohydrates, it does not completely remove the carbohydrate,minimizing risks of exposing potentially protease-sensitive or antigenicpolypeptide sites.

The half-life of lysosomal enzyme β-glucuronidase is known to increasemore than tenfold after sequential treatment with periodate and sodiumborohydride (Houba et al. (1996) Bioconjug. Chem. 7(5):606-11; Stahl etal (1976) PNAS 73:4045-4049; Achord et al. (1977) Pediat, Res.11:816-822; Achord et al. (1978) Cell 15(1):269-78). Similarly, ricinhas been treated with a mixture of periodate and sodium cyanoborohydride(Thorpe et al. (1985) Eur, J. Biochem. 147:197). After injection intorats, the fraction of ricin adsorbed by the liver decreased from 40%(untreated ricin) to 20% (modified ricin) of the injected dose withchemical treatment. In contrast the amount of ricin in the bloodincreased from 20% (untreated ricin) to 45% (treated ricin). Thus, thetreated ricin enjoyed a wider tissue distribution and longer half-lifein the circulation.

A β-glucuronidase construct (or other glycoprotein) coupled to atargeting moiety of the invention when deglycosylated or modified bysequential treatment with periodate and sodium borohydride should enjoya similar (e.g. more than twofold, more than fourfold, or more thantenfold) increase in halflife while still retaining a high affinity forthe cation-independent M6P receptor, permitting targeting of theconstruct to the lysosome of all cell types that possess this receptor.The construct is also predicted to cross the blood brain barrierefficiently. In contrast, if a β-glucuronidase preparation that relieson M6P for lysosomal targeting is deglycosylated or treated withperiodate and sodium borohydride, it will enjoy an elevated serumhalf-life but will be unable to target the lysosome since the M6Ptargeting signal will have been modified by the treatment.

Carbohydrate modification by sequential treatment with periodate andsodium borohydride can be performed as follows: Purified GILT-GUS isincubated with 40 mM NaIO₄ in 50 mM sodium acetate pH 4.5 for 2 hours at4° C. The reaction is stopped by addition of excess ethylene glycol andunreacted reagents are removed by passing the reaction mix over SephadexG-25M equilibrated with PBS pH 7.5. This treatment is followed byincubation with 40 mM NaBH₄ in PBS at pH 7.5 and 37° C. for three hoursand then for one hour at 4° C. Passing the reaction mixture over aSephadex G-25M column eluted with PBS at pH 7.5 terminates the reaction.

Another protocol for periodate and sodium borohydride treatment isdescribed in Hickman et al. (1974) BBRC 57:55-61. The purified proteinis dialyzed into 0.01M sodium phosphate pH 6.0, 0.15 M NaCl. Sodiumperiodate is added to a final concentration of 0.01M and the reactionproceeds at 4° C. in the dark for at least six hours. Treatment ofβ-hexosaminidase with periodate under these conditions is sufficient toprevent uptake of the protein by fibroblasts; uptake is normallydependent on M6P moieties on the β-hexosaminidase with the M6P receptoron the fibroblast cell surface. Thus, periodate oxidation modifies M6Psufficiently to abolish its ability to interact with the M6P receptor.

Alternatively, the carbohydrate can be modified by treatment withperiodate and cyanoborohydride in a one step reaction as disclosed inThorpe et al. (1985) Eur. J. Biochem. 147:197-206.

The presence of carbohydrate in a partially deglycosylated protein or aprotein with a modified glycosylation pattern should shield potentialpolypeptide epitopes that might be uncovered by complete absence ofglycosylation. In the event that a therapeutic protein does provoke animmune response, immunosuppressive therapies can be used in conjunctionwith the therapeutic protein (Brooks (1999) Molecular Genetics andMetabolism 68:268-275). For example, it has been reported that about 15%of Gaucher disease patients treated with alglucerase developed immuneresponses (Beutler, et al., in The Metabolic and Molecular Bases ofInherited Disease, 8^(th) ed. (2001), Scriver et al., eds., pp.3635-3668). Fortunately, many (82/142) of the patients that producedantibody against alglucerase became tolerized by the normal treatmentregimen (Rosenberg et al., (1999) Blood 93:2081-2088). Thus, to benefitthe small minority of patients who may develop an immune response, apatient receiving a therapeutic protein also receives animmunosuppressive therapy in some embodiments of the invention.

Testing

To verify that a protein is underglycosylated, it can be tested byexposure to ConA. An underglycosylated protein is expected todemonstrate reduced binding to ConA-sepharose when compared to thecorresponding fully glycosylated protein.

An actually underglycosylated protein can also be resolved by SDS-PAGEand compared to the corresponding fully-glycosylated protein. Forexample, chemically deglycosylated GUS-GILT can be compared to untreated(glycosylated) GUS-GILT and to enzymatically deglycosylated GUS-GILTprepared with PNGase A. The underglycosylated protein is expected tohave a greater mobility in SDS-PAGE when compared to the fullyglycosylated protein.

Underglycosylated targeted therapeutic proteins display uptake that isdependent on the targeting domain. Underglycosylated proteins shoulddisplay reduced uptake (and, preferably, substantially no uptake) thatis dependent on mannose or M6P. These properties can be experimentallyverified in cell uptake experiments.

For example, a GUS-GILT protein synthesized in mammalian cells andsubsequently treated with periodate and borohydride can be tested forfunctional deglycosylation by testing M6P-dependent andmannose-dependent uptake. To demonstrate that M6P-dependent uptake hasbeen reduced, uptake assays are performed using GM4668 fibroblasts. Inthe absence of competitor, treated and untreated enzyme will eachdisplay significant uptake. The presence of excess IGF-II substantiallyreduces uptake of treated and untreated enzyme, although untreatedenzyme retains residual uptake via a M6P-dependent pathway. Excess M6Preduces the uptake of untreated enzyme, but is substantially lesseffective at reducing the uptake of functionally deglycosylated protein.For treated and untreated enzymes, the simultaneous presence of bothcompetitors should substantially abolish uptake.

Uptake assays to assess mannose-dependent uptake are performed usingJ774-E cells, a mouse macrophage-like cell line bearing mannosereceptors but few, if any, M6P receptors (Diment et al. (1987) J.Leukocyte Biol. 42:485-490). The cells are cultured in DMEM, lowglucose, supplemented with 10% FBS, 4 mM glutamine, and antibiotic,antimycotic solution (Sigma, A-5955). Uptake assays with these cells areperformed in a manner identical to assays performed with fibroblasts. Inthe presence of excess M6P and IGF-II, which will eliminate uptake dueto any residual M6P/IGF-II receptor, fully glycosylated enzyme willdisplay significant uptake due to interaction with the mannose receptor.Underglycosylated enzyme is expected to display substantially reduceduptake under these conditions. The mannose receptor-dependent uptake offully glycosylated enzyme can be competed by the addition of excess (100μg/mL) mannan.

Pharmacokinetics of deglycosylated GUS-GILT can be determined by givingintravenous injections of 20,000 enzyme units to groups of three MPSVIImice per timepoint. For each timepoint 50 μL of blood is assayed forenzyme activity.

INCORPORATION BY REFERENCE

The disclosure of each of the patent documents, scientific publications,and Protein Data Bank records disclosed herein, and U.S. ProvisionalApplication No. 60/250,446, filed Nov. 30, 2000; U.S. ProvisionalApplication 60/287,531, filed Apr. 30, 2001; U.S. ProvisionalApplication 60/290,281, filed May 11, 2001; U.S. Provisional Application60/304,609, filed Jul. 10, 2001; U.S. Provisional Application No.60/329,461, filed Oct. 15, 2001; International Patent Application SerialNo. PCT/US01/44935, filed Nov. 30, 2001; U.S. Provisional ApplicationNo. 60/351,276, filed Jan. 23, 2002; U.S. Ser. Nos. 10/136,841 and10/136,639, filed Apr. 30, 2002, U.S. Ser. No. 60/384,452, filed May 29,2002; U.S. Ser. No. 60/386,019, filed Jun. 5, 2002; and U.S. Ser. No.60/408,816, filed Sep. 6, 2002, are incorporated by reference into thisapplication in their entirety.

We claim:
 1. A nucleic acid encoding a targeted therapeutic fusionprotein comprising: a lysosomal enzyme; and a lysosomal targeting domainthat binds human cation-independent mannose-6-phosphate receptor in amannose-6-phosphate-independent manner; wherein the lysosomal targetingdomain comprises a mutein of mature human IGF-II having an amino acidsequence at least 70% identical to mature human IGF-II (SEQ ID NO:8). 2.The nucleic acid of claim 1, wherein the IGF-II moiety comprises adeletion or replacement of amino acids 1-7 of mature human IGF-II. 3.The nucleic acid of claim 1, wherein the IGF-II moiety comprises: (a)amino acids 8-67 of mature human IGF-II; (b) amino acids 1-61 of maturehuman IGF-II; or (c) amino acids 8-61 of mature human IGF-II.
 4. Thenucleic acid of claim 1, wherein the IGF-II moiety comprises amino acids8-67 of mature human IGF-II.
 5. The nucleic acid of claim 1, wherein thevariant of human IGF-II comprises a deletion or replacement of aminoacids 62-67 of mature human IGF-II.
 6. The nucleic acid of claim 1,wherein the variant of human IGF-II comprises a deletion or areplacement of amino acids 29-40 of mature human IGF-II.
 7. The nucleicacid of claim 1, wherein the variant of human IGF-II comprises adeletion or a replacement of amino acids 1-7 and a substitution ofTyr27Leu of mature human IGF-II.
 8. The nucleic acid of claim 1, whereinthe variant of human IGF-II comprises one or more substitutions of aminoacid residues Tyr27 with Leu, Leu43 with Val, and/or Ser36 with Phe ofmature human IGF-II.
 9. The nucleic acid of claim 1, wherein the variantof human IGF-II comprises amino acids 41-61 of human IGF-II and a muteinof amino acids 8-28 of human IGF-II, the mutein differing from humanIGF-II at a position selected from the group consisting of amino acid 9,amino acid 19, amino acid 26, and amino acid 27 of mature human IGF-II.10. The nucleic acid of claim 1, wherein the mutein of mature humanIGF-II comprises amino acids 48-55 of mature human IGF-II.
 11. Thenucleic acid of claim 1, wherein the mutein of mature human IGF-IIcomprises, at corresponding positions, at least three amino acidsselected from the group consisting of amino acids 8, 48, 49, 50, 54, and55 of mature human IGF-II.
 12. The nucleic acid of claim 1, wherein theamino acid sequence comprises, at positions corresponding to positions54 and 55 of mature human IGF-II, amino acids each of which areuncharged or negatively charged at pH 7.4.
 13. The nucleic acid of claim1, wherein the mutein of mature human IGF-II comprises the amino acidsequence phenylalanine-arginine-serine.
 14. The nucleic acid of claim 1,wherein the mutein of mature human IGF-II comprises amino acids 8-28 and41-61 of mature human IGF-II.
 15. The nucleic acid of claim 1, whereinthe fusion protein further comprises a bridge between the lysosomalenzyme and the lysosomal targeting moiety.
 16. The nucleic acid of claim15, wherein the bridge comprises the amino acid sequence of GAP.
 17. Thenucleic acid of claim 1, wherein the lysosomal enzyme is selected fromthe group consisting of acid-α1,4-glucosidase; β-galactosidase;β-hexosaminidase A; β-hexosaminidase B; α-galactosidase A;glucocerebrosidase; arylsulfatase B; galactosylceramidase; acidsphingomyelinase; acid ceramidase; acid lipase; α-L-iduronidase;iduronate sulfatase; heparan N-sulfatase; α-N-acetylglucosaminidase;acetyl-CoA-glucosaminide acetyltransferase;N-acetylglucosamine-6-sulfatase; galactosamine-6-sulfatase;arylsulfatase B; β-glucuronidase; α-mannosidase; β-mannosidase;α-L-fucosidase; N-aspartyl-β-glucosaminidase; α-neuraminidase; lysosomalprotective protein; α-N-acetyl- galactosaminidase;N-acetylglucosamine-1-phosphotransferase; and palmitoyl-proteinthioesterase.
 18. A gene therapy vector comprising a nucleic acidencoding a targeted therapeutic protein of claim
 1. 19. The gene therapyvector of claim 18 that is a viral vector.
 20. A cell comprising thenucleic acid of claim
 1. 21. A method of treating a patient with alysosomal storage disease, the method comprising administering to thepatient a nucleic acid encoding a therapeutic fusion protein as set outin claim
 1. 22. The method of claim 21, wherein the lysosomal storagedisease is selected from the group consisting of Pompe Disease; TaySachs disease; Sandhoff disease; Fabry disease; Gaucher disease;Mucopolysaccharidosis (MPS) I; MPS II, MPS IVA, MPSIVB, MPS VII;α-Mannosidosis; Wolman disease and Sanfilippo B.
 23. A method oftreating a patient with a lysosomal storage disease, comprisingadministering to the patient a gene therapy vector as set out in claim18.
 24. The method of claim 23, wherein the lysosomal storage disease isselected from the group consisting of Pompe Disease; Tay Sachs disease;Sandhoff disease; Fabry disease; Gaucher disease; Mucopolysaccharidosis(MPS) I; MPS II, MPS IVA, MPSIVB, MPS VII; α-Mannosidosis; Wolmandisease and Sanfilippo B.